Prevention and treatment of synucleinopathic disease

ABSTRACT

The invention provides improved agents and methods for treatment of diseases associated with synucleinopathic diseases, including Lewy bodies of alpha-synuclein in the brain of a patient. Such methods entail administering agents that induce a beneficial immunogenic response against the Lewy body. The methods are particularly useful for prophylactic and therapeutic treatment of Parkinson&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Application No. 60/423,012, filed Nov. 1, 2002, which is incorporated byreference herein for all purposes.

BACKGROUND OF THE INVENTION

Alpha-synuclein (alphaSN) brain pathology is a conspicuous feature ofseveral neurodegenerative diseases, including Parkinson's disease (PD),dementia with Lewy bodies (DLB), the Lewy body variant of Alzheimer'sdisease (LBVAD), multiple systems atrophy (MSA), and neurodegenerationwith brain iron accumulation type-1 (NBIA-1). Common to all of thesediseases, termed synucleinopathies, are proteinaceous insolubleinclusions in the neurons and the glia which are composed primarily ofalphaSN.

Lewy bodies and Lewy neurites are intraneuronal inclusions which arecomposed primarily of alphaSN. Lewy bodies and Lewy neurites are theneuropathological hallmarks Parkinson's disease (PD). PD and othersynucleinopathic diseases have been collectively referred to as Lewybody disease (LBD). LBD is characterized by degeneration of thedopaminergic system, motor alterations, cognitive impairment, andformation of Lewy bodies (LBs). (McKeith et al., Clinical andpathological diagnosis of dementia with Lewy bodies (DLB): Report of theCDLB International Workshop, Neurology (1996) 47:1113-24). Other LBDsinclude diffuse Lewy body disease (DLBD), Lewy body variant ofAlzheimer's disease (LBVAD), combined PD and Alzheimer's disease (AD),and multiple systems atrophy.

Disorders with LBs continue to be a common cause for movement disordersand cognitive deterioration in the aging population (Galasko et al.,Clinical-neuropathological correlations in Alzheimer's disease andrelated dementias. Arch. Neurol. (1994) 51:888-95). Although theirincidence continues to increase creating a serious public healthproblem, to date these disorders are neither curable nor preventable andunderstanding the causes and pathogenesis of PD is critical towardsdeveloping new treatments (Tanner et al., Epidemiology of Parkinson'sdisease and akinetic syndromes, Curr. Opin. Neurol. (2000) 13:427-30).The cause for PD is controversial and multiple factors have beenproposed to play a role, including various neurotoxins and geneticsusceptibility factors.

In recent years, new hope for understanding the pathogenesis of PD hasemerged. Specifically, several studies have shown that the synapticprotein alpha-SN plays a central role in PD pathogenesis since: (1) thisprotein accumulates in LBs (Spillantini et al., Nature (1997)388:839-40; Takeda et al., AM. J. Pathol. (1998) 152:367-72; Wakabayashiet al., Neurosci. Lett. (1997) 239:45-8), (2) mutations in the alpha-SNgene co-segregate with rare familial forms of parkinsonism (Kruger etal., Nature Gen. (1998) 18:106-8; Polymeropoulos M H, et al., Science(1997) 276:2045-7) and, (3) its overexpression in transgenic mice(Masliah et al., Science (2000) 287.1265-9) and Drosophila (Feany etal., Nature (2000) 404:394-8) mimics several pathological aspects of PD.Thus, the fact that accumulation of alpha-SN in the brain is associatedwith similar morphological and neurological alterations in species asdiverse as humans, mice, and flies suggests that this moleculecontributes to the development of PD.

An alpha-SN fragment, previously determined to be a constituent of ADamyloid plaques, was termed the non-amyloid-beta (non-Aβ) component ofAD amyloid (NAC) (Iwai A., Biochim. Biophys. Acta (2000) 1502:95-109);Masliah et al., AM. J. Pathol (1996) 148:201-10; Ueda et al., Proc.Natl. Acad. Sci. USA (1993) 90:11282-6). Although the precise functionof NAC is not known, it may play a critical role in synaptic events,such as neural plasticity during development, and learning anddegeneration of nerve terminals under pathological conditions in LBD,AD, and other disorders (Hasimoto et al., Alpha-Synuclein in Lewy bodydisease and Alzheimer's disease, Brain Pathol (1999) 9:707-20; Masliah,et al., (2000).

AD, PD, and dementia with Lewy bodies (DLB) are the most commonly foundneurodegenerative disorders in the elderly. Although their incidencecontinues to increase, creating a serious public health problem, to datethese disorders are neither curable nor preventable. Recentepidemiological studies have demonstrated a close clinical relationshipbetween AD and PD, as about 30% of Alzheimer's patients also have PD.Compared to the rest of the aging population, patients with AD are thusmore likely to develop concomitant PD. Furthermore, PD patients thatbecome demented usually have developed classical AD. Although eachneurodegenerative disease appears to have a predilection for specificbrain regions and cell populations, resulting in distinct pathologicalfeatures, PD, AD, DLB and LBD also share common pathological hallmarks.Patients with familial AD, Down syndrome, or sporadic AD develop LBs onthe amygdala, which are the classical neuropathological hallmarks of PD.Additionally, each disease is associated with the degeneration ofneurons, interneuronal synaptic connections and eventually cell death,the depletion of neurotransmitters, and abnormal accumulation ofmisfolded proteins, the precursors of which participate in normalcentral nervous system function. Biochemical studies have confirmed thelink between AD, PD and DLB.

The neuritic plaques that are the classic pathological hallmark of ADcontain beta-amyloid (Aβ) peptide and non-beta amyloid component (NAC)peptide. AP is derived from a larger precursor protein termed amyloidprecursor protein (APP). NAC is derived from a larger precursor proteintermed the non-beta amyloid component of APP, now more commonly referredto as alpha-SN. NAC comprises amino acid residues 60-87 or 61-95 ofalpha-SN. Both Aβ and NAC were first identified in amyloid plaques asproteolytic fragments of their respective full-length proteins, forwhich the full-length cDNAs were identified and cloned.

Alpha-SN is part of a large family of proteins including beta- andgamma-synuclein and synoretin. Alpha-SN is expressed in the normal stateassociated with synapses and is believed to play a role in neuralplasticity, learning and memory. Mutations in human (h) alpha-SN thatenhance the aggregation of alpha-SN have been identified (Ala30Pro andAla53Thr) and are associated with rare forms of autosomal dominant formsof PD. The mechanism by which these mutations increase the propensity ofalpha-SN to aggregate are unknown.

Despite the fact that a number of mutations can be found in APP andalpha-SN in the population, most cases of AD and PD are sporadic. Themost frequent sporadic forms of these diseases are associated with anabnormal accumulation of Aβ and alpha-SN, respectively. However, thereasons for over accumulation of these proteins is unknown. Aβ issecreted from neurons and accumulates in extracellular amyloid plaques.Additionally Aβ can be detected inside neurons. Alpha-SN accumulates inintraneuronal inclusions called LBs. Although the two proteins aretypically found together in extracellular neuritic AD plaques, they arealso occasionally found together in intracellular inclusions.

The mechanisms by which alpha-SN accumulation leads to neurodegenerationand the characteristics symptoms of PD are unclear. However, identifyingthe role of factors promoting and/or blocking alpha-SN aggregation iscritical for the understanding of LBD pathogenesis and development ofnovel treatments for its associated disorders. Research for identifyingtreatments has been directed toward searching for compounds that reducealpha-SN aggregation (Hashimoto, et al.) or testing growth factors thatwill promote the regeneration and/or survival of dopaminergic neurons,which are the cells primarily affected (Djaldetti et al., New therapiesfor Parkinson's disease, J. Neurol (2001) 248:357-62; Kirik et al.,Long-term rAAV-mediated gene transfer of GDNF in the rat Parkinson'smodel: intrastriatal but not intranigral transduction promotesfunctional regeneration in the lesioned nigrostriatal system, J.Neurosci (2000) 20:4686-4700). Recent studies in a transgenic mousemodel of AD have shown that antibodies against Aβ 1-42 facilitate andstimulate the removal of amyloid from the brain, improve AD-likepathology and resulting in improve cognitive performance (Schenk et al.,Immunization with amyloid-β attenuates Alzheimer-disease-like pathologyin PDAPP mouse, Nature (1999) 408:173-177; Morgan et al., A-beta peptidevaccination prevents memory loss in an animal model of Alzheimer'sdisease, Nature (2000) 408:982-985; Janus et al., A-beta peptideimmunization reduces behavioral impairment and plaques in a model ofAlzheimer's disease, Nature (2000) 408:979-82). In contrast to theextracellular amyloid plaques found in the brains of Alzheimer'spatients, Lewy bodies are intracellular, and antibodies do not typicallyenter the cell.

Surprisingly, given the intracellular nature of LBs in brain tissue, theinventors have succeeded in reducing the number of inclusions intransgenic mice immunized with synuclein. The present invention isdirected inter alia to treatment of PD and other diseases associatedwith LBs by administration of synuclein, fragments of synuclein,antigens that mimic synuclein or fragments thereof, or antibodies tocertain epitopes of synuclein to a patient under conditions thatgenerate a beneficial immune response in the patient. The inventors havealso surprisingly succeeded in reducing the number of inclusions intransgenic mice immunized with Aβ. The present invention is directedinter alia to treatment of PD and other diseases associated with LBs byadministration of Aβ, fragments of Aβ, antigens that mimic Aβ orfragments thereof, or antibodies to certain epitopes of Aβ to a patientunder conditions that generate a beneficial immune response in thepatient. The invention thus fulfills a longstanding need for therapeuticregimes for preventing or ameliorating the neuropathology and, in somepatients, the cognitive impairment associated with PD and other diseasesassociated with LBs.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides methods of preventing or treatinga disease characterized by Lewy bodies or alpha-SN aggregation in thebrain. Such methods entail, inducing an immunogenic response againstalpha-SN. Such induction may be achieved by active administration of animmunogen or passive by administration of an antibody or a derivative ofan antibody to synuclein. In some methods, the patient is asymptomatic.In some methods, the patient has the disease and is asymptomatic. Insome methods the patient has a risk factor for the disease and isasymptomatic. In some methods, the disease is Parkinson's disease. Insome methods, the disease is Parkinson's disease, and the administeringthe agent improves motor characteristics of the patient. In somemethods, the disease is Parkinson's disease administering the agentprevents deterioration of motor characteristics of the patient. In somemethods, the patient is free of Alzheimer's disease.

For treatment of patients suffering from Lewy bodies or alpha-SNaggregation in the brain, one treatment regime entails administering adose of alpha-SN or an active fragment thereof to the patient to inducethe immune response. In some methods the alpha-SN or an active fragmentthereof is administered in multiple doses over a period of at least sixmonths. The alpha-SN or an active fragment thereof can be administered,for example, peripherally, intraperitoneally, orally, subcutaneously,intracranially, intramuscularly, topically, intranasally orintravenously. In some methods, the alpha-SN or an active fragmentthereof is administered with an adjuvant that enhances the immuneresponse to the alpha-SN or an active fragment thereof. In some methods,the immunogenic response comprises antibodies to alpha-SN or an activefragment thereof.

In some methods, the agent is amino acids 35-65 of alpha-SN. In somemethods, the agent comprises amino acids 130-140 of alpha-SN and hasfewer than 40 amino acids total. In some methods, the C-terminal aminoacids of the agent are the C-terminal amino acid of alpha-SN. In some ofthe above methods, the alpha-SN or active fragment is linked to acarrier molecule to form a conjugate. In some of the above methods, thealpha-SN or active fragment is linked to a carrier at the N-terminus ofthe alpha-SN or active fragment.

For treatment of patients suffering from Lewy bodies or alpha-SNaggregation in the brain, one treatment regime entails administering adose of an antibody to alpha-SN or an active fragment thereof to thepatient to induce the immune response. The antibody used may be human,humanized, chimeric, or polyclonal and can be monoclonal or polyclonal.In some methods the isotype of the antibody is a human IgG1. In somemethods, the antibody is prepared from a human immunized with alpha-SNpeptide and the human can be the patient to be treated with antibody. Insome methods, the antibody binds to the outer surface of neuronal cellshaving Lewy bodies thereby dissipating the Lewy bodies. In some methods,the antibody is internalized within neuronal cells having Lewy bodiesthereby dissipating the Lewy bodies.

In some methods, the antibody is administered with a pharmaceuticalcarrier as a pharmaceutical composition. In some methods, antibody isadministered at a dosage of 0.0001 to 100 mg/kg, preferably, at least 1mg/kg body weight antibody. In some methods the antibody is administeredin multiple doses over a prolonged period, for example, at least sixmonths. In some methods antibodies may be administered as a sustainedrelease composition. The antibody can be administered, for example,peripherally, intraperitoneally, orally, subcutaneously, intracranially,intramuscularly, topically, intranasally or intravenously. In somemethods, the patient is monitored for level of administered antibody inthe blood of the patient.

In some methods, the antibody is administered by administering apolynucleotide encoding at least one antibody chain to the patient. Thepolynucleotide is expressed to produce the antibody chain in thepatient. Optionally, the polynucleotide encodes heavy and light chainsof the antibody and the polynucleotide is expressed to produce the heavyand light chains in the patient.

This invention further provides pharmaceutical compositions comprisingan antibody to alpha-SN and a pharmaceutically acceptable carrier.

In another aspect, the invention provides methods of preventing ortreating a disease characterized by Lewy bodies or alpha-SN aggregationin the brain comprising administering an agent that induces animmunogenic response against alpha-SN, and further comprisingadministering of a second agent that induces an immunogenic responseagainst Aβ to the patient. In some methods, the agent is Aβ or an activefragment thereof. In some methods, the agent is an antibody to Aβ.

In another aspect, the invention provides methods of preventing ortreating a disease characterized by Lewy bodies or alpha-SN aggregationin the brain comprising administering an agent that induces animmunogenic response against Aβ to a patient. In some methods, the agentis Aβ or an active fragment thereof. In some methods, the agent is anantibody to Aβ. In some methods the disease is Parkinson's disease. Insome methods, the patient is free of Alzheimer's disease and has no riskfactors thereof. In some methods, further comprise monitoring a sign orsymptom of Parkinson's disease in the patient. In some methods, thedisease is Parkinson's disease and administering the agent results inimprovement in a sign or symptom of Parkinson's disease.

This invention further provides pharmaceutical compositions comprisingan agent effective to induce an immunogenic response against a componentof a Lewy body in a patient, such as described above, and apharmaceutically acceptable adjuvant. In some compounds, the agent isalpha-SN or an active fragment, for example, NAC. In some compounds theagent is 6CHC-1 or an active fragment. The invention also providespharmaceutical compositions comprising an antibody specific for acomponent of a Lewy body.

In another aspect, the invention provides for methods of screening anantibody for activity in preventing or treating a disease associatedwith Lewy bodies. Such methods entail, contacting a neuronal cellexpressing synuclein with the antibody. Then one determines whether thecontacting reduces synuclein deposits in the cells compared with acontrol cells not contacted with the antibody.

In another aspect, the invention provides for methods of screening anantibody for activity in treating or preventing a Lewy body disease inthe brain of a patient. Such methods entail contacting the antibody witha polypeptide comprising at least five contiguous amino acids ofalpha-SN. Then one determines whether the antibody specifically binds tothe polypeptide, specific binding providing an indication that theantibody has activity in treating the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of alpha-SN (SEQ ID: 1) inalignment with two NAC amino acid sequences, SEQ ID NO: 2 and SEQ ID NO:3, respectively.

FIG. 2 shows immunohistostained brain sections from nontransgenic mice(panels A, E, and I), alpha-SN transgenic mice immunized with adjuvantalone (panels B, F, J), and alpha-SN transgenic mice immunized withalpha-SN which developed low titers (panels C, G, and K) and high titers(panels D, H, and I) of antibodies to alpha-SN. Sections were subjectedto staining with an anti-alpha-synuclein antibody to detect synucleinlevels (panels A-D), an anti-IgG antibody to determine total IgG levelspresent in the section (panels E-H), and for Glial Fibrillary AcidicProtein (GFAP), a marker of astroglial cells.

FIG. 3 shows the effects of anti-mSYN polyclonal antibody on synucleinaggregation in transfected GT1-7 cells as seen by light microscopy.

FIG. 4 is a Western blot of synuclein levels in the cytoplasm (C) andmembranes (P) of GT1-7 α-syn cells treated with preimmune sera and with67-10 antibody at a concentration of (1:50) for 48 hours prior toanalysis.

FIG. 5 shows the results of studies of the effect of Aβ1-42 immunizationamyloid deposition in the brains of nontransgenic, SYN, APP and SYN/APPtransgenic mice. The detectable amyloid levels seen in APP and SYN/APPmice are reduced by Aβ1-42 immunization.

FIG. 6 shows the results of studies of the effect of Aβ1-42 immunizationupon synuclein inclusion formation in the brains of nontransgenic, SYN,APP and SYN/APP transgenic mice. Synuclein inclusions detected in SYNand SYN/APP mice are reduced by Aβ1-42 immunization.

FIG. 7 shows direct and indirect mechanisms by which antibodies blockalpha-SN aggregation.

DEFINITIONS

The term “substantial identity” means that two peptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 65 percent sequence identity, preferably atleast 80 or 90 percent sequence identity, more preferably at least 95percent sequence identity or more (e.g., 99 percent sequence identity orhigher). Preferably, residue positions which are not identical differ byconservative amino acid substitutions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra). One example of algorithm that is suitable fordetermining percent sequence identity and sequence similarity is theBLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(NCBI) website. Typically, default program parameters can be used toperform the sequence comparison, although customized parameters can alsobe used. For amino acid sequences, the BLASTP program uses as defaults aword length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89, 10915(1989)).

For purposes of classifying amino acids substitutions as conservative ornon-conservative, amino acids are grouped as follows: Group I(hydrophobic sidechains): norleucine, met, ala, val, leu, ile; Group II(neutral hydrophilic side chains): cys, ser, thr; Group III (acidic sidechains): asp, glu; Group IV (basic side chains): asn, gln, his, lys,arg; Group V (residues influencing chain orientation): gly, pro; andGroup VI (aromatic side chains): trp, tyr, phe. Conservativesubstitutions involve substitutions between amino acids in the sameclass. Non-conservative substitutions constitute exchanging a member ofone of these classes for a member of another.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained.

The phrase that a molecule “specifically binds” to a target refers to abinding reaction which is determinative of the presence of the moleculein the presence of a heterogeneous population of other biologics. Thus,under designated immunoassay conditions, a specified molecule bindspreferentially to a particular target and does not bind in a significantamount to other biologics present in the sample. Specific binding of anantibody to a target under such conditions requires the antibody beselected for its specificity to the target. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See, e.g., Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity. Specific binding betweentwo entities means an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹ M⁻¹, or10¹⁰ M⁻¹. Affinities greater than 10⁸ M⁻¹ are preferred.

The term “antibody” or “immunoglobulin” is used to include intactantibodies and binding fragments thereof. Typically, fragments competewith the intact antibody from which they were derived for specificbinding to an antigen fragment including separate heavy chains, lightchains Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes one or moreimmunoglobulin chains that are chemically conjugated to, or expressedas, fusion proteins with other proteins. The term “antibody” alsoincludes bispecific antibody. A bispecific or bifunctional antibody isan artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553(1992).

APP⁶⁹⁵, APP⁷⁵¹, and APP⁷⁷⁰ refer, respectively, to the 695, 751, and 770amino acid residue long polypeptides encoded by the human APP gene. SeeKang et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525(1988); and Kitaguchi et al., Nature 331, 530 (1988). Amino acids withinthe human amyloid precursor protein (APP) are assigned numbers accordingto the sequence of the APP770 isoform. Terms such as Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43 refer to an Aβ peptide containing amino acid residues1-39, 1-40, 1-41, 1-42 and 1-43.

An “antigen” is an entity to which an antibody specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which B and/or T cells respond. B-cell epitopes can be formedboth from contiguous amino acids or noncontiguous amino acids juxtaposedby tertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, and moreusually, at least 5 or 8-10 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies thatrecognize the same epitope can be identified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen. T-cells recognize continuous epitopes ofabout nine amino acids for CD8 cells or about 13-15 amino acids for CD4cells. T cells that recognize the epitope can be identified by in vitroassays that measure antigen-dependent proliferation, as determined by³H-thymidine incorporation by primed T cells in response to an epitope(Burke et al., J. Inf. Dis. 170, 1110-19 (1994)), by antigen-dependentkilling (cytotoxic T lymphocyte assay, Tigges et al., J. Immunol. 156,3901-3910) or by cytokine secretion.

The term “immunological” or “immune” response is the development of abeneficial humoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst an amyloid peptide in a recipient patient. Such a response canbe an active response induced by administration of immunogen or apassive response induced by administration of antibody or primedT-cells. A cellular immune response is elicited by the presentation ofpolypeptide epitopes in association with Class I or Class II MHCmolecules to activate antigen-specific CD4⁺ T helper cells and/or CD8⁺cytotoxic T cells. The response may also involve activation ofmonocytes, macrophages, NK cells, basophils, dendritic cells,astrocytes, microglia cells, eosinophils or other components of innateimmunity. The presence of a cell-mediated immunological response can bedetermined by proliferation assays (CD4⁺ T cells) or CTL (cytotoxic Tlymphocyte) assays (see Burke, supra; Tigges, supra). The relativecontributions of humoral and cellular responses to the protective ortherapeutic effect of an immunogen can be distinguished by separatelyisolating antibodies and T-cells from an immunized syngeneic animal andmeasuring protective or therapeutic effect in a second subject.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

The term “all-D” refers to peptides having ≧75%, ≧80%, ≧85%, ≧90%, ≧95%,and 100% D-configuration amino acids.

The term “naked polynucleotide” refers to a polynucleotide not complexedwith colloidal materials. Naked polynucleotides are sometimes cloned ina plasmid vector.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments the immune response to the antigen,but when administered alone does not generate an immune response to theantigen. Adjuvants can augment an immune response by several mechanismsincluding lymphocyte recruitment, stimulation of B and/or T cells, andstimulation of macrophages.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

Competition between antibodies is determined by an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as alpha-SN. Numerous types ofcompetitive binding assays are known, for example: solid phase direct orindirect radioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242-253 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay, solid phase direct labeledsandwich assay (see Harlow and Lane, Antibodies, A Laboratory Manual,Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabelled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50 or 75%.

The term “symptom” or “clinical symptom” refers to a subjective evidenceof a disease, such as altered gait, as perceived by the patient. A“sign” refers to objective evidence of a disease as observed by aphysician.

Compositions or methods “comprising” one or more recited elements mayinclude other elements not specifically recited. For example, acomposition that comprises alpha-SN peptide encompasses both an isolatedalpha-SN peptide and alpha-SN peptide as a component of a largerpolypeptide sequence.

DETAILED DESCRIPTION OF THE INVENTION I. General

The invention provides methods of preventing or treating severaldiseases and conditions characterized by presence of deposits ofalpha-SN peptide aggregated to an insoluble mass in the brain of apatient, in the form of Lewy bodies. Such diseases are collectivelyreferred to as Lewy Body diseases (LBD) and include Parkinson's disease(PD). Such diseases are characterized by aggregates of alpha-SN thathave a β-pleated sheet structure and stain with thioflavin-S andCongo-red (see Hasimoto, Ibid). The invention provides methods ofpreventing or treating such diseases using an agent that can generate animmunogenic response to alpha-SN. The immunogenic response acts toprevent formation of, or clear, synuclein deposits within cells in thebrain. Although an understanding of mechanism is not essential forpractice of the invention, the immunogenic response may induce clearingas a result of antibodies to synuclein that are internalized withincells and/or which interact with the membrane of such cells and therebyinterfere with aggregation of synuclein. In some methods, the clearingresponse can be effected at least in part by Fc receptor mediatedphagocytosis. Immunization with synuclein can reduce synucleinaccumulation at synapses in the brain. Although an understanding ofmechanism is not essential for practice of the invention, this resultcan be explained by antibodies to synuclein being taken up by synapticvesicles.

Optionally, agents generating an immunogenic response against alpha-SNcan be used in combination with agents that generate an immunogenicresponse to Aβ. The immunogenic response is useful in clearing depositsof Aβ in individuals having such deposits (e.g., individuals having bothAlzheimer's and Parkinson's disease); however, the immunogenic responsealso has activity in clearing synuclein deposits. Thus, the presentinvention uses such agents alone, or in combination with agentsgenerating an immunogenic response to alpha-SN in individuals with LBDbut who are not suffering or at risk of developing Alzheimer's disease.

II. Agents Generating an Immunogenic Response Against Alpha Synuclein

An immunogenic response can be active, as when an immunogen isadministered to induce antibodies reactive with alpha-SN in a patient,or passive, as when an antibody is administered that itself binds toalpha-SN in a patient.

1. Agents Inducing Active Immune Response

Therapeutic agents induce an immunogenic response specifically directedto certain epitopes within the alpha-SN peptide. Preferred agents arethe alpha-SN peptide itself and fragments thereof. Variants of suchfragments, analogs and mimetics of natural alpha-SN peptide that induceand/or cross-react with antibodies to the preferred epitopes of alpha-SNpeptide can also be used.

Alpha synuclein was originally identified in human brains as theprecursor protein of the non-β-amyloid component of (NAC) of AD plaques.(Ueda et al., Proc. Natl. Acad. Sci. U.S.A. 90 (23):11282-11286 (1993).Alpha-SN, also termed the precursor of the non-Aβ component of ADamyloid (NACP), is a peptide of 140 amino acids. Alpha-SN has the aminoacid sequence:

(SEQ ID NO: 1) MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA(Uéda et al., Ibid.; GenBank accession  number: P37840).

The non-Aβ component of AD amyloid (NAC) is derived from alpha-SN. NAC,a highly hydrophobic domain within alpha synuclein, is a peptideconsisting of at least 28 amino acids residues (residues 60-87) (SEQ IDNO: 3) and optionally 35 amino acid residues (residues 61-95) (SEQ IDNO: 2). See FIG. 1. NAC displays a tendency to form a beta-sheetstructure (Iwai, et al., Biochemistry, 34:10139-10145). Jensen et al.have reported NAC has the amino acid sequence:

(SEQ ID NO: 2) EQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFV (Jensen et al., Biochem. J. 310 (Pt 1): 91-94(1995); GenBankaccession number S56746).

Uéda et al. have reported NAC has the acid sequence:

KEQVTNVGGAVVTGVTAVAQKTVEGAGS (SEQ ID NO: 3) (Uéda et al., PNAS USA90: 11282-11286 (1993).

Disaggregated alpha-SN or fragments thereof, including NAC, meansmonomeric peptide units. Disaggregated alpha-SN or fragments thereof aregenerally soluble, and are capable of self-aggregating to form solubleoligomers. Oligomers of alpha-SN and fragments thereof are usuallysoluble and exist predominantly as alpha-helices. Monomeric alpha-SN maybe prepared in vitro by dissolving lyophilized peptide in neat DMSO withsonication. The resulting solution is centrifuged to remove anyinsoluble particulates. Aggregated alpha-SN or fragments thereof,including NAC, means oligomers of alpha-SN or fragments thereof whichhave associate into insoluble beta-sheet assemblies. Aggregated alpha-SNor fragments thereof, including NAC, means also means fibrillarpolymers. Fibrils are usually insoluble. Some antibodies bind eithersoluble alpha-SN or fragments thereof or aggregated alpha-SN orfragments thereof. Some antibodies bind both soluble and aggregatedalpha-SN or fragments thereof.

Alpha-SN, the principal component of the Lewy bodies characteristic ofPD, and epitopic fragments thereof, such as, for example, NAC, orfragments other than NAC, can be used to induce an immunogenic response.Preferably such fragments comprise four or more amino acids of alpha-SNor analog thereof. Some active fragments include an epitope at or nearthe C-terminus of alpha-SN (e.g., within amino acids 70-140, 100-140,120-140, 130-140, or 135-140). In some active fragments, the C terminalresidue of the epitope is the C-terminal residue of alpha-SN. Othercomponents of Lewy bodies, for example, synphilin-1, Parkin, ubiquitin,neurofilament, beta-crystallin, and epitopic fragments thereof can alsobe used to induce an immunogenic response.

Unless otherwise indicated, reference to alpha-SN includes the naturalhuman amino acid sequences indicated above as well as analogs includingallelic, species and induced variants, full-length forms and immunogenicfragments thereof. Analogs typically differ from naturally occurringpeptides at one, two or a few positions, often by virtue of conservativesubstitutions. Analogs typically exhibit at least 80 or 90% sequenceidentity with natural peptides. Some analogs also include unnaturalamino acids or modifications of N or C terminal amino acids at one, twoor a few positions. For example, the natural glutamic acid residue atposition 139 of alpha-SN can be replaced with iso-aspartic acid.Examples of unnatural amino acids are D, alpha, alpha-disubstitutedamino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,gamma-carboxyglutamate, epsilon-N,N,N-trimethyllysine,epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,omega-N-methylarginine, β-alanine, ornithine, norleucine, norvaline,hydroxyproline, thyroxine, gamma-amino butyric acid, homoserine,citrulline, and isoaspartic acid. Therapeutic agents also includeanalogs of alpha-SN fragments. Some therapeutic agents of the inventionare all-D peptides, e.g., all-D alpha-SN or all-D NAC, and of all-Dpeptide analogs. Fragments and analogs can be screened for prophylacticor therapeutic efficacy in transgenic animal models in comparison withuntreated or placebo controls as described below.

Alpha-SN, its fragments, and analogs can be synthesized by solid phasepeptide synthesis or recombinant expression, or can be obtained fromnatural sources. Automatic peptide synthesizers are commerciallyavailable from numerous suppliers, such as Applied Biosystems, FosterCity, Calif. Recombinant expression can be in bacteria, such as E. coli,yeast, insect cells or mammalian cells. Procedures for recombinantexpression are described by Sambrook et al., Molecular Cloning: ALaboratory Manual (C.S.H.P. Press, NY 2d ed., 1989). Some forms ofalpha-SN peptide are also available commercially, for example, at BACHEMand American Peptide Company, Inc.

Therapeutic agents also include longer polypeptides that include, forexample, an active fragment of alpha-SN peptide, together with otheramino acids. For example, preferred agents include fusion proteinscomprising a segment of alpha-SN fused to a heterologous amino acidsequence that induces a helper T-cell response against the heterologousamino acid sequence and thereby a B-cell response against the alpha-SNsegment. Such polypeptides can be screened for prophylactic ortherapeutic efficacy in animal models in comparison with untreated orplacebo controls as described below. The alpha-SN peptide, analog,active fragment or other polypeptide can be administered in associatedor multimeric form or in dissociated form therapeutic agents alsoinclude multimers of monomeric immunogenic agents. The therapeuticagents of the invention may include polylysine sequences.

In a further variation, an immunogenic peptide, such as a fragment ofalpha-SN, can be presented by a virus or bacteria as part of animmunogenic composition. A nucleic acid encoding the immunogenic peptideis incorporated into a genome or episome of the virus or bacteria.Optionally, the nucleic acid is incorporated in such a manner that theimmunogenic peptide is expressed as a secreted protein or as a fusionprotein with an outer surface protein of a virus or a transmembraneprotein of bacteria so that the peptide is displayed. Viruses orbacteria used in such methods should be nonpathogenic or attenuated.Suitable viruses include adenovirus, HSV, Venezuelan equine encephalitisvirus and other alpha viruses, vesicular stomatitis virus, and otherrhabdo viruses, vaccinia and fowl pox. Suitable bacteria includeSalmonella and Shigella. Fusion of an immunogenic peptide to HBsAg ofHBV is particularly suitable.

Therapeutic agents also include peptides and other compounds that do notnecessarily have a significant amino acid sequence similarity withalpha-SN but nevertheless serve as mimetics of alpha-SN and induce asimilar immune response. For example, any peptides and proteins formingbeta-pleated sheets can be screened for suitability. Anti-idiotypicantibodies against monoclonal antibodies to alpha-SN or other Lewy bodycomponents can also be used. Such anti-Id antibodies mimic the antigenand generate an immune response to it (see Essential Immunology, Roited., Blackwell Scientific Publications, Palo Alto, Calif. 6th ed., p.181). Agents other than alpha-SN should induce an immunogenic responseagainst one or more of the preferred segments of alpha-SN listed above(e.g., NAC). Preferably, such agents induce an immunogenic response thatis specifically directed to one of these segments without being directedto other segments of alpha-SN.

Random libraries of peptides or other compounds can also be screened forsuitability. Combinatorial libraries can be produced for many types ofcompounds that can be synthesized in a step-by-step fashion. Suchcompounds include polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines and oligocarbamates. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in Affymax, WO 95/12608, Affymax, WO 93/06121, ColumbiaUniversity, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO95/30642 (each of which is incorporated herein by reference for allpurposes). Peptide libraries can also be generated by phage displaymethods. See, e.g., Devlin, W0 91/18980.

Combinatorial libraries and other compounds are initially screened forsuitability by determining their capacity to bind to antibodies orlymphocytes (B or T) known to be specific for alpha-SN or other Lewybody components. For example, initial screens can be performed with anypolyclonal sera or monoclonal antibody to alpha-SN or a fragmentthereof. Compounds can then be screened for binding to a specificepitope within alpha-SN (e.g., an epitope within NAC). Compounds can betested by the same procedures described for mapping antibody epitopespecificities. Compounds identified by such screens are then furtheranalyzed for capacity to induce antibodies or reactive lymphocytes toalpha-SN or fragments thereof. For example, multiple dilutions of seracan be tested on microtiter plates that have been precoated withalpha-SN or a fragment thereof and a standard ELISA can be performed totest for reactive antibodies to alpha-SN or the fragment. Compounds canthen be tested for prophylactic and therapeutic efficacy in transgenicanimals predisposed to a disease associated with the presence of Lewybody, as described in the Examples. Such animals include, for example,transgenic mice over expressing alpha-SN or mutant thereof (e.g.,alanine to threonine substitution at position 53) as described, e.g., inWO 98/59050, Masliah, et al., Science 287: 1265-1269 (2000), and van derPutter, et al., J. Neuroscience 20: 6025-6029 (2000), or such transgenicmice that also over express APP or a mutant thereof. Particularlypreferred are such synuclein transgenic mice bearing a 717 mutation ofAPP described by Games et al., Nature 373, 523 (1995) and mice bearing a670/671 Swedish mutation of APP such as described by McConlogue et al.,U.S. Pat. No. 5,612,486 and Hsiao et al., Science 274, 99 (1996);Staufenbiel et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292 (1997);Sturchler-Pierrat et al., Proc. Natl. Acad. Sci. USA 94, 13287-13292(1997); Borchelt et al., Neuron 19, 939-945 (1997)). Examples of suchsynuclein/APP transgenic animals are provided in WO 01/60794. Additionalanimal models of PD include 6-hydroxydopamine, rotenone, and1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal models (M.Flint Beal, Nature Reviews Neuroscience 2:325-334 (2001)). The samescreening approach can be used on other potential analogs of alpha-SNand longer peptides including fragments of alpha-SN, described above andother Lewy body components and analog or fragments thereof.

2. Agents for Passive Immune Response

Therapeutic agents of the invention also include antibodies thatspecifically bind to alpha-SN or other components of Lewy bodies.Antibodies immunoreactive for alpha-SN are known (see, for example,Arima, et al., Brian Res. 808: 93-100 (1998); Crowther et al.,Neuroscience Lett. 292: 128-130 (2000); Spillantini, et al. Nature 388:839-840 (1997). Such antibodies can be monoclonal or polyclonal. Somesuch antibodies bind specifically to insoluble aggregates of alpha-SNwithout binding to the soluble monomeric form. Some bind specifically tothe soluble monomeric form without binding to the insoluble aggregatedform. Some bind to both aggregated and soluble monomeric forms. Somesuch antibodies bind to a naturally occurring short form of alpha-SN(e.g., NAC) without binding to a naturally occurring full lengthalpha-SN. Some antibodies bind to a long form without binding to a shortform. Some antibodies bind to alpha-SN without binding to othercomponents of LBs. Antibodies used in therapeutic methods usually havean intact constant region or at least sufficient of the constant regionto interact with an Fc receptor. Human isotype IgG1 is preferred becauseof it having highest affinity of human isotypes for the FcRI receptor onphagocytic cells. Bispecific Fab fragments can also be used, in whichone arm of the antibody has specificity for alpha-SN, and the other foran Fc receptor. Some antibodies bind to alpha-SN with a binding affinitygreater than or equal to about 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹.

Polyclonal sera typically contain mixed populations of antibodiesbinding to several epitopes along the length of alpha-SN. However,polyclonal sera can be specific to a particular segment of alpha-SN,such as NAC. Monoclonal antibodies bind to a specific epitope withinalpha-SN that can be a conformational or nonconformational epitope.Prophylactic and therapeutic efficacy of antibodies can be tested usingthe transgenic animal model procedures described in the Examples.Preferred monoclonal antibodies bind to an epitope within NAC. In somemethods, multiple monoclonal antibodies having binding specificities todifferent epitopes are used. Such antibodies can be administeredsequentially or simultaneously. Antibodies to Lewy body components otherthan alpha-SN can also be used. For example, antibodies can be directedto neurofilament, ubiquitin, or synphilin. Therapeutic agents alsoinclude antibodies raised against analogs of alpha-SN and fragmentsthereof. Some therapeutic agents of the invention are all-D peptides,e.g., all-D alpha-SN or all-D NAC.

When an antibody is said to bind to an epitope within specifiedresidues, such as alpha-SN 1-5, for example, what is meant is that theantibody specifically binds to a polypeptide containing the specifiedresidues (i.e., alpha-SN 1-5 in this an example). Such an antibody doesnot necessarily contact every residue within alpha-SN 1-5. Nor doesevery single amino acid substitution or deletion with in alpha-SN1-5necessarily significantly affect binding affinity. Epitope specificityof an antibody can be determined, for example, by forming a phagedisplay library in which different members display differentsubsequences of alpha-SN. The phage display library is then selected formembers specifically binding to an antibody under test. A family ofsequences is isolated. Typically, such a family contains a common coresequence, and varying lengths of flanking sequences in differentmembers. The shortest core sequence showing specific binding to theantibody defines the epitope bound by the antibody. Antibodies can alsobe tested for epitope specificity in a competition assay with anantibody whose epitope specificity has already been determined.

Some antibodies of the invention specifically binds to an epitope withinNAC. Some antibodies specifically binds to an epitope within a22-kilodalton glycosylated form of synuclein, e.g., P22-synuclein (H.Shimura et al., Science 2001 Jul. 13:293(5528):224-5). Some antibodiesbinds to an epitope at or near the C-terminus of alpha-SN (e.g., withinamino acids 70-140, 100-140, 120-140, 130-140 or 135-140. Someantibodies bind to an epitope in which the C-terminal residue of theepitope is the C-terminal residue of alpha-SN. In some methods, theantibody specifically binds to NAC without binding to full lengthalpha-SN.

Monoclonal or polyclonal antibodies that specifically bind to apreferred segment of alpha-SN without binding to other regions ofalpha-SN have a number of advantages relative to monoclonal antibodiesbinding to other regions or polyclonal sera to intact alpha-SN. First,for equal mass dosages, dosages of antibodies that specifically bind topreferred segments contain a higher molar dosage of antibodies effectivein clearing amyloid plaques. Second, antibodies specifically binding topreferred segments can induce a clearing response against LBs withoutinducing a clearing response against intact alpha-SN, thereby reducingthe potential for side effects.

i. General Characteristics of Immunoglobulins

The basic antibody structural unit is known to comprise a tetramer ofsubunits. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,Ch. 7 (incorporated by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.,1987 and 1991); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); orChothia et al., Nature 342:878-883 (1989).

ii. Production of Nonhuman Antibodies

Chimeric and humanized antibodies have the same or similar bindingspecificity and affinity as a mouse or other nonhuman antibody thatprovides the starting material for construction of a chimeric orhumanized antibody. Chimeric antibodies are antibodies whose light andheavy chain genes have been constructed, typically by geneticengineering, from immunoglobulin gene segments belonging to differentspecies. For example, the variable (V) segments of the genes from amouse monoclonal antibody may be joined to human constant (C) segments,such as IgG1 and IgG4. Human isotype IgG1 is preferred. In some methods,the isotype of the antibody is human IgG1. IgM antibodies can also beused in some methods. A typical chimeric antibody is thus a hybridprotein consisting of the V or antigen-binding domain from a mouseantibody and the C or effector domain from a human antibody.

Humanized antibodies have variable region framework residuessubstantially from a human antibody (termed an acceptor antibody) andcomplementarity determining regions substantially from a mouse-antibody,(referred to as the donor immunoglobulin). See, Queen et al., Proc.Natl. Acad. Sci. USA 86:10029-10033 (1989), WO 90/07861, U.S. Pat. No.5,693,762, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,585,089, U.S. Pat.No. 5,530,101, and Winter, U.S. Pat. No. 5,225,539 (each of which isincorporated by reference in its entirety for all purposes). Theconstant region(s), if present, are also substantially or entirely froma human immunoglobulin. The human variable domains are usually chosenfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable region domains from which theCDRs were derived. The heavy and light chain variable region frameworkresidues can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Carter et al., WO 92/22653. Certain aminoacids from the human variable region framework residues are selected forsubstitution based on their possible influence on CDR conformationand/or binding to antigen. Investigation of such possible influences isby modeling, examination of the characteristics of the amino acids atparticular locations, or empirical observation of the effects ofsubstitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid should usually be substituted bythe equivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,(2) is adjacent to a CDR region,(3) otherwise interacts with a CDR region (e.g. is within about 6 A of aCDR region), or(4) participates in the VL-VH interface.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of the mouse donor antibody or from the equivalentpositions of more typical human immunoglobulins. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. The variable regionframeworks of humanized immunoglobulins usually show at least 85%sequence identity to a human variable region framework sequence orconsensus of such sequences.

iv. Human Antibodies

Human antibodies against alpha-SN are provided by a variety oftechniques described below. Some human antibodies are selected bycompetitive binding experiments, or otherwise, to have the same epitopespecificity as a particular mouse antibody, such as one of the mousemonoclonals described in Example XI. Human antibodies can also bescreened for a particular epitope specificity by using only a fragmentof alpha-SN as the immunogen, and/or by screening antibodies against acollection of deletion mutants of alpha-SN. Human antibodies preferablyhave isotype specificity human IgG1.

(1) Trioma Methodology

The basic approach and an exemplary cell fusion partner, SPAZ-4, for usein this approach have been described by Oestberg et al., Hybridoma2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman etal., U.S. Pat. No. 4,634,666 (each of which is incorporated by referencein its entirety for all purposes). The antibody-producing cell linesobtained by this method are called triomas, because they are descendedfrom three cells-two human and one mouse. Initially, a mouse myelomaline is fused with a human B-lymphocyte to obtain anon-antibody-producing xenogeneic hybrid cell, such as the SPAZ-4 cellline described by Oestberg, supra. The xenogeneic cell is then fusedwith an immunized human B-lymphocyte to obtain an antibody-producingtrioma cell line. Triomas have been found to produce antibody morestably than ordinary hybridomas made from human cells.

The immunized B-lymphocytes are obtained from the blood, spleen, lymphnodes or bone marrow of a human donor. If antibodies against a specificantigen or epitope are desired, it is preferable to use that antigen orepitope thereof for immunization. Immunization can be either in vivo orin vitro. For in vivo immunization, B cells are typically isolated froma human immunized with alpha-SN, a fragment thereof, larger polypeptidecontaining alpha-SN or fragment, or an anti-idiotypic antibody to anantibody to alpha-SN. In some methods, B cells are isolated from thesame patient who is ultimately to be administered antibody therapy. Forin vitro immunization, B-lymphocytes are typically exposed to antigenfor a period of 7-14 days in a media such as RPMI-1640 (see Engleman,supra) supplemented with 10% human plasma.

The immunized B-lymphocytes are fused to a xenogeneic hybrid cell suchas SPAZ-4 by well known methods. For example, the cells are treated with40-50% polyethylene glycol of MW 1000-4000, at about 37 degrees C., forabout 5-10 min. Cells are separated from the fusion mixture andpropagated in media selective for the desired hybrids (e.g., HAT or AH).Clones secreting antibodies having the required binding specificity areidentified by assaying the trioma culture medium for the ability to bindto alpha-SN or a fragment thereof. Triomas producing human antibodieshaving the desired specificity are subcloned by the limiting dilutiontechnique and grown in vitro in culture medium. The trioma cell linesobtained are then tested for the ability to bind alpha-SN or a fragmentthereof.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into standard mammalian, bacterial or yeast celllines.

(2) Transgenic Non-Human Mammals

Human antibodies against alpha-SN can also be produced from non-humantransgenic mammals having transgenes encoding at least a segment of thehuman immunoglobulin locus. Usually, the endogenous immunoglobulin locusof such transgenic mammals is functionally inactivated. Preferably, thesegment of the human immunoglobulin locus includes unrearrangedsequences of heavy and light chain components. Both inactivation ofendogenous immunoglobulin genes and introduction of exogenousimmunoglobulin genes can be achieved by targeted homologousrecombination, or by introduction of YAC chromosomes. The transgenicmammals resulting from this process are capable of functionallyrearranging the immunoglobulin component sequences, and expressing arepertoire of antibodies of various isotypes encoded by humanimmunoglobulin genes, without expressing endogenous immunoglobulingenes. The production and properties of mammals having these propertiesare described in detail by, e.g., Lonberg et al., WO93/1222, U.S. Pat.No. 5,877,397, U.S. Pat. No. 5,874,299, U.S. Pat. No. 5,814,318, U.S.Pat. No. 5,789,650, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,661,016,U.S. Pat. No. 5,633,425, U.S. Pat. No. 5,625,126, U.S. Pat. No.5,569,825, U.S. Pat. No. 5,545,806, Nature 148, 1547-1553 (1994), NatureBiotechnology 14, 826 (1996), Kucherlapati, WO 91/10741 (each of whichis incorporated by reference in its entirety for all purposes).Transgenic mice are particularly suitable. Anti-alpha-SN antibodies areobtained by immunizing a transgenic nonhuman mammal, such as describedby Lonberg or Kucherlapati, supra, with alpha-SN or a fragment thereof.Monoclonal antibodies are prepared by, e.g., fusing B-cells from suchmammals to suitable myeloma cell lines using conventionalKohler-Milstein technology. Human polyclonal antibodies can also beprovided in the form of serum from humans immunized with an immunogenicagent. Optionally, such polyclonal antibodies can be concentrated byaffinity purification using alpha-SN or other amyloid peptide as anaffinity reagent.

(3) Phage Display Methods

A further approach for obtaining human anti-alpha-SN antibodies is toscreen a DNA library from human B cells according to the generalprotocol outlined by Huse et al., Science 246:1275-1281 (1989). Asdescribed for trioma methodology, such B cells can be obtained from ahuman immunized with alpha-SN, fragments, longer polypeptides containingalpha-SN or fragments or anti-idiotypic antibodies. Optionally, such Bcells are obtained from a patient who is ultimately to receive antibodytreatment. Antibodies binding to alpha-SN or a fragment thereof areselected. Sequences encoding such antibodies (or binding fragments) arethen cloned and amplified. The protocol described by Huse is renderedmore efficient in combination with phage-display technology. See, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat.No. 5,877,218, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,858,657, U.S.Pat. No. 5,837,242, U.S. Pat. No. 5,733,743 and U.S. Pat. No. 5,565,332(each of which is incorporated by reference in its entirety for allpurposes). In these methods, libraries of phage are produced in whichmembers display different antibodies on their outer surfaces. Antibodiesare usually displayed as Fv or Fab fragments. Phage displayingantibodies with a desired specificity are selected by affinityenrichment to an alpha-SN peptide or fragment thereof.

In a variation of the phage-display method, human antibodies having thebinding specificity of a selected murine antibody can be produced. SeeWinter, WO 92/20791. In this method, either the heavy or light chainvariable region of the selected murine antibody is used as a startingmaterial. If, for example, a light chain variable region is selected asthe starting material, a phage library is constructed in which membersdisplay the same light chain variable region (i.e., the murine startingmaterial) and a different heavy chain variable region. The heavy chainvariable regions are obtained from a library of rearranged human heavychain variable regions. A phage showing strong specific binding foralpha-SN (e.g., at least 10⁸ and preferably at least 10⁹ M⁻¹) isselected. The human heavy chain variable region from this phage thenserves as a starting material for constructing a further phage library.In this library, each phage displays the same heavy chain variableregion (i.e., the region identified from the first display library) anda different light chain variable region. The light chain variableregions are obtained from a library of rearranged human variable lightchain regions. Again, phage showing strong specific binding for alpha-SNare selected. These phage display the variable regions of completelyhuman anti-alpha-SN antibodies. These antibodies usually have the sameor similar epitope specificity as the murine starting material.

v. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized, orhuman antibodies can be linked to at least a portion of a human constantregion. The choice of constant region depends, in part, whetherantibody-dependent complement and/or cellular mediated toxicity isdesired. For example, isotopes IgG1 and IgG3 have complement activityand isotypes IgG2 and IgG4 do not. Choice of isotype can also affectpassage of antibody into the brain. Human isotype IgG1 is preferred.Light chain constant regions can be lambda or kappa. Antibodies can beexpressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′ F(ab′)2, and Fv, or assingle chain antibodies in which heavy and light chain variable domainsare linked through a spacer.

vi. Expression of Recombinant Antibodies

Chimeric, humanized and human antibodies are typically produced byrecombinant expression. Recombinant polynucleotide constructs typicallyinclude an expression control sequence operably linked to the codingsequences of antibody chains, including naturally associated orheterologous promoter regions. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g.,ampicillin-resistance or hygromycin-resistance, to permit detection ofthose cells transformed with the desired DNA sequences.

E. coli is one prokaryotic host particularly useful for cloning the DNAsequences of the present invention. Microbes, such as yeast are alsouseful for expression. Saccharomyces is a preferred yeast host, withsuitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, NY, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines, various COS celllines, HeLa cells, L cells, human embryonic kidney cell, and myelomacell lines. Preferably, the cells are nonhuman. Expression vectors forthese cells can include expression control sequences, such as an originof replication, a promoter, an enhancer (Queen et al., Immunol. Rev.89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from endogenous genes, cytomegalovirus,SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J.Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. No. 5,741,957, U.S. Pat. No. 5,304,489, U.S. Pat. No.5,849,992). Suitable transgenes include coding sequences for lightand/or heavy chains in operable linkage with a promoter and enhancerfrom a mammary gland specific gene, such as casein or betalactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment,electroporation, lipofection, biolistics or viral-based transfection canbe used for other cellular hosts. Other methods used to transformmammalian cells include the use of polybrene, protoplast fusion,liposomes, electroporation, and microinjection (see generally, Sambrooket al. supra). For production of transgenic animals, transgenes can bemicroinjected into fertilized oocytes, or can be incorporated into thegenome of embryonic stem cells, and the nuclei of such cells transferredinto enucleated oocytes.

Once expressed, antibodies can be purified according to standardprocedures of the art, including HPLC purification, columnchromatography, gel electrophoresis and the like (see generally, Scopes,Protein Purification (Springer-Verlag, NY, 1982)).

3. Conjugates

Some agents for inducing an immune response contain the appropriateepitope for inducing an immune response against LBs but are too small tobe immunogenic. In this situation, a peptide immunogen can be linked toa suitable carrier molecule to form a conjugate which helps elicit animmune response. Suitable carriers include serum albumins, keyholelimpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin,tetanus toxoid, or a toxoid from other pathogenic bacteria, such asdiphtheria, E. coli, cholera, or H. pylori, or an attenuated toxinderivative. T cell epitopes are also suitable carrier molecules. Someconjugates can be formed by linking agents of the invention to animmunostimulatory polymer molecule (e.g., tripalmitoyl-S-glycerinecysteine (Pam₃Cys), mannan (a manose polymer), or glucan (a beta 1→2polymer)), cytokines (e.g., IL-1, IL-1 alpha and beta peptides, IL-2,gamma-INF, IL-10, GM-CSF), and chemokines (e.g., MIP1alpha and beta, andRANTES). Immunogenic agents can also be linked to peptides that enhancetransport across tissues, as described in O'Mahony, WO 97/17613 and WO97/17614. Immunogens may be linked to the carries with or with outspacers amino acids (e.g., gly-gly).

Some conjugates can be formed by linking agents of the invention to atleast one T cell epitope. Some T cell epitopes are promiscuous whileother T cell epitopes are universal. Promiscuous T cell epitopes arecapable of enhancing the induction of T cell immunity in a wide varietyof subjects displaying various HLA types. In contrast to promiscuous Tcell epitopes, universal T cell epitopes are capable of enhancing theinduction of T cell immunity in a large percentage, e.g., at least 75%,of subjects displaying various HLA molecules encoded by different HLA-DRalleles.

A large number of naturally occurring T-cell epitopes exist, such as,tetanus toxoid (e.g., the P2 and P30 epitopes), Hepatitis B surfaceantigen, pertussis, toxoid, measles virus F protein, Chlamydiatrachomitis major outer membrane protein, diphtheria toxoid, Plasmodiumfalciparum circumsporozite T, Plasmodium falciparum CS antigen,Schistosoma mansoni triose phosphate isomersae, Escherichia coli TraT,and Influenza virus hemagluttinin (HA). The immunogenic peptides of theinvention can also be conjugated to the T-cell epitopes described inSinigaglia F. et al., Nature, 336:778-780 (1988); Chicz R. M. et al., J.Exp. Med., 178:27-47 (1993); Hammer J. et al., Cell 74:197-203 (1993);Falk K. et al., Immunogenetics, 39:230-242 (1994); WO 98/23635; and,Southwood S. et al. J. Immunology, 160:3363-3373 (1998) (each of whichis incorporated herein by reference for all purposes). Further examplesinclude:

Influenza Hemagluttinin: HA₃₀₇₋₃₁₉ PKYVKQNTLKLAT (SEQ ID NO: 4)

Malaria CS: T3 epitope EKKIAKMEKASSVFNV (SEQ ID NO: 5)

Hepatitis B surface antigen: HBsAg₁₉₋₂₈ FFLLTRILTI (SEQ ID NO: 6)

Heat Shock Protein 65: hsp65₁₅₃₋₁₇₁ DQSIGDLIAEAMDKVGNEG (SEQ ID NO: 7)

bacille Calmette-Guerin QVHFQPLPPAVVKL (SEQ ID NO: 8)

Tetanus toxoid: TT₈₃₀₋₈₄₄ QYIKANSKFIGITEL (SEQ ID NO: 9)

Tetanus toxoid: TT₉₄₇₋₉₆₇ FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 10)

HIV gp120 T1: KQIINMWQEVGKAMYA (SEQ ID NO: 11)

Alternatively, the conjugates can be formed by linking agents of theinvention to at least one artificial T-cell epitope capable of binding alarge proportion of MHC Class II molecules., such as the pan DR epitope(“PADRE”). PADRE is described in U.S. Pat. No. 5,736,142, WO 95/07707,and Alexander J et al., Immunity, 1:751-761 (1994) (each of which isincorporated herein by reference for all purposes). A preferred PADREpeptide is AKXVAAWTLKAAA (SEQ ID NO: 12), (common residues bolded)wherein X is preferably cyclohexylalanine, tyrosine or phenylalanine,with cyclohexylalanine being most preferred.

Immunogenic agents can be linked to carriers by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) (if the peptide lacks a sulfhydryl group, this can be provided byaddition of a cysteine residue). These reagents create a disulfidelinkage between themselves and peptide cysteine resides on one proteinand an amide linkage through the epsilon-amino on a lysine, or otherfree amino group in other amino acids. A variety of suchdisulfide/amide-forming agents are described by Immun. Rev. 62, 185(1982). Other bifunctional coupling agents form a thioether rather thana disulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenicity can be improved through the addition of spacer residues(e.g., Gly-Gly) between the T_(h) epitope and the peptide immunogen ofthe invention. In addition to physically separating the T_(h) epitopefrom the B cell epitope (i.e., the peptide immunogen), the glycineresidues can disrupt any artificial secondary structures created by thejoining of the T_(h) epitope with the peptide immunogen, and therebyeliminate interference between the T and/or B cell responses. Theconformational separation between the helper epitope and the antibodyeliciting domain thus permits more efficient interactions between thepresented immunogen and the appropriate T_(h) and B cells.

To enhance the induction of T cell immunity in a large percentage ofsubjects displaying various HLA types to an agent of the presentinvention, a mixture of conjugates with different T_(h) cell epitopescan be prepared. The mixture may contain a mixture of at least twoconjugates with different T_(h) cell epitopes, a mixture of at leastthree conjugates with different T_(h) cell epitopes, or a mixture of atleast four conjugates with different T_(h) cell epitopes. The mixturemay be administered with an adjuvant.

Immunogenic peptides can also be expressed as fusion proteins withcarriers (i.e., heterologous peptides). The immunogenic peptide can belinked at its amino terminus, its carboxyl terminus, or both to acarrier. Optionally, multiple repeats of the immunogenic peptide can bepresent in the fusion protein. Optionally, an immunogenic peptide can belinked to multiple copies of a heterologous peptide, for example, atboth the N and C termini of the peptide. Some carrier peptides serve toinduce a helper T-cell response against the carrier peptide. The inducedhelper T-cells in turn induce a B-cell response against the immunogenicpeptide linked to the carrier peptide.

Some agents of the invention comprise a fusion protein in which anN-terminal fragment of alpha-SN is linked at its C-terminus to a carrierpeptide. In such agents, the N-terminal residue of the fragment ofalpha-SN constitutes the N-terminal residue of the fusion protein.Accordingly, such fusion proteins are effective in inducing antibodiesthat bind to an epitope that requires the N-terminal residue of alpha-SNto be in free form. Some agents of the invention comprise a plurality ofrepeats of NAC linked at the C-terminus to one or more copy of a carrierpeptide. Some fusion proteins comprise different segments of alpha-SN intandem.

In some fusion proteins, NAC is fused at its N-terminal end to aheterologous carrier peptide. NAC can be used with C-terminal fusions.Some fusion proteins comprise a heterologous peptide linked to theN-terminus or C-terminus of NAC, which is in turn linked to one or moreadditional NAC segments of alpha-SN in tandem.

Some examples of fusion proteins suitable for use in the invention areshown below. Some of these fusion proteins comprise segments of alpha-SNlinked to tetanus toxoid epitopes such as described in U.S. Pat. No.5,196,512, EP 378,881 and EP 427,347. Some fusion proteins comprisesegments of alpha-SN linked to at least one PADRE Some heterologouspeptides are promiscuous T-cell epitopes, while other heterologouspeptides are universal T-cell epitopes. In some methods, the agent foradministration is simply a single fusion protein with an alpha-SNsegment linked to a heterologous segment in linear configuration. Thetherapeutic agents of the invention may be represented using a formula.For example, in some methods, the agent is multimer of fusion proteinsrepresented by the formula 2^(x), in which x is an integer from 1-5.Preferably x is 1, 2, or 3, with 2 being most preferred. When x is two,such a multimer has four fusion proteins linked in a preferredconfiguration referred to as MAP4 (see U.S. Pat. No. 5,229,490).

The MAP4 configuration is shown below, where branched structures areproduced by initiating peptide synthesis at both the N terminal and sidechain amines of lysine. Depending upon the number of times lysine isincorporated into the sequence and allowed to branch, the resultingstructure will present multiple N termini. In this example, fouridentical N termini have been produced on the branched lysine-containingcore. Such multiplicity greatly enhances the responsiveness of cognate Bcells.

Z refers to the NAC peptide, a fragment of the NAC peptide, or otheractive fragment of alpha-SN as described in section I. 2 above. Z mayrepresent more than one active fragment, for example:

Z=alpha-SN 60-72 (NAC region) peptide=NH2-KEQVTNVCGGAVVT-COOH (SEQ IDNO: 13)Z=alpha-SN 73-84 (NAC region) peptide=NH2-GVTAVAQKTVECG-COOH (SEQ ID NO:14)Z=alpha-SN 102-112 peptide=NH2-C-amino-heptanoic acid-KNEEGAPCQEG-COOH(SEQ ID NO: 15)alpha-SN 128-140 peptide

Other examples of fusion proteins include:

Z-Tetanus toxoid 830-844 in a MAP4 configuration:

Z-QYIKANSKFIGITEL (SEQ ID NO: 16)Z-Tetanus toxoid 947-967 in a MAP4 configuration:

Z-FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 17)Z-Tetanus toxoid₈₃₀₋₈₄₄ in a MAP4 configuration:

Z-QYIKANSKFIGITEL (SEQ ID NO: 18)

Z-Tetanus toxoid₈₃₀₋₈₄₄+Tetanus toxoid₉₄₇₋₉₆₇ in a linear configuration:

(SEQ ID NO: 19) Z-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE

PADRE peptide (all in linear configurations), wherein X is preferablycyclohexylalanine, tyrosine or phenylalanine, with cyclohexylalaninebeing most preferred-Z:

AKXVAAWTLKAAA-Z (SEQ ID NO: 20)3Z-PADRE peptide:

Z-Z-Z-AKXVAAWTLKAAA (SEQ ID NO: 21)

Further examples of fusion proteins include:

(SEQ ID NO: 22) AKXVAAWTLKAAA-Z-Z-Z-Z (SEQ ID NO: 23) Z-AKXVAAWTLKAAA(SEQ ID NO: 24) Z-ISQAVHAAHAEINEAGR (SEQ ID NO: 25) PKYVKQNTLKLAT-Z-Z-Z (SEQ ID NO: 26) Z-PKYVKQNTLKLAT-Z  (SEQ ID NO: 27) Z-Z-Z-PKYVKQNTLKLAT (SEQ ID NO: 28) Z-Z-PKYVKQNTLKLAT  (SEQ ID NO: 29)Z-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE-Z-Z-Z-Z-QYIKANSKFIGITEL-FNN FTVSFWLRVPKVSASHLE (SEQ ID NO: 30) Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-ZZ-QYIKANSKFIGITEL (SEQ ID NO: 31) on a 2 branched resin:

The same or similar carrier proteins and methods of linkage can be usedfor generating immunogens to be used in generation of antibodies againstalpha-SN for use in passive immunization. For example, alpha-SN or afragment linked to a carrier can be administered to a laboratory animalin the production of monoclonal antibodies to alpha-SN.

4. Nucleic Acid Encoding Therapeutic Agents

Immune responses against Lewy bodies can also be induced byadministration of nucleic acids encoding segments of alpha-SN peptide,and fragments thereof, other peptide immunogens, or antibodies and theircomponent chains used for passive immunization. Such nucleic acids canbe DNA or RNA. A nucleic acid segment encoding an immunogen is typicallylinked to regulatory elements, such as a promoter and enhancer thatallow expression of the DNA segment in the intended target cells of apatient. For expression in blood cells, as is desirable for induction ofan immune response, promoter and enhancer elements from light or heavychain immunoglobulin genes or the CMV major intermediate early promoterand enhancer are suitable to direct expression. The linked regulatoryelements and coding sequences are often cloned into a vector. Foradministration of double-chain antibodies, the two chains can be clonedin the same or separate vectors. The nucleic acid encoding therapeuticagents of the invention may also encode at least one T cell epitope. Thedisclosures herein which relates to the use of adjuvants and the use ofapply mutatis mutandis to their use with the nucleic acid encodingtherapeutic agents of the present invention.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3,102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou etal., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519(1996)), Venezuelan equine encephalitis virus (see U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (see WO96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy 6,325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, NucleicAcids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. No. 5,208,036, U.S. Pat. No. 5,264,618, U.S. Pat.No. 5,279,833, and U.S. Pat. No. 5,283,185. Vectors and DNA encoding animmunogen can also be adsorbed to or associated with particulatecarriers, examples of which include polymethyl methacrylate polymers andpolylactides and poly(lactide-co-glycolides), (see, e.g., McGee et al.,J. Micro Encap. 1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346). Such vectorscan further include facilitating agents such as bupivacine (see e.g.,U.S. Pat. No. 5,593,970). DNA can also be administered using a gene gun.See Xiao & Brandsma, supra. The DNA encoding an immunogen isprecipitated onto the surface of microscopic metal beads. Themicroprojectiles are accelerated with a shock wave or expanding heliumgas, and penetrate tissues to a depth of several cell layers. Forexample, The Accel™ Gene Delivery Device manufactured by Agacetus, Inc.Middleton, Wis. is suitable. Alternatively, naked DNA can pass throughskin into the blood stream simply by spotting the DNA onto skin withchemical or mechanical irritation (see WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, and tissue biopsy) or universaldonor hematopoietic stem cells, followed by reimplantation of the cellsinto a patient, usually after selection for cells which haveincorporated the vector.

III. Agents for Inducing Immunogenic Response Against Aβ

Aβ, also known as β-amyloid peptide, or A4 peptide (see U.S. Pat. No.4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun. 120, 1131(1984)), is a peptide of 39-43 amino acids, which is the principalcomponent of characteristic plaques of Alzheimer's disease. Aβ isgenerated by processing of a larger protein APP by two enzymes, termed βand γ secretases (see Hardy, TINS 20, 154 (1997)). Known mutations inAPP associated with Alzheimer's disease occur proximate to the site of βor γ secretase, or within Aβ. For example, position 717 is proximate tothe site of γ-secretase cleavage of APP in its processing to Aβ, andpositions 670/671 are proximate to the site of β-secretase cleavage. Itis believed that the mutations cause AD by interacting with the cleavagereactions by which Aβ is formed so as to increase the amount of the42/43 amino acid form of Aβ generated.

Aβ has the unusual property that it can fix and activate both classicaland alternate complement cascades. In particular, it binds to C1q andultimately to C3bi. This association facilitates binding to macrophagesleading to activation of B cells. In addition, C3bi breaks down furtherand then binds to CR2 on B cells in a T cell dependent manner leading toa 10,000 increase in activation of these cells. This mechanism causes Aβto generate an immune response in excess of that of other antigens.

Aβ has several natural occurring forms. The human forms of Aβ arereferred to as Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43. The sequences of thesepeptides and their relationship to the APP precursor are illustrated byFIG. 1 of Hardy et al., TINS 20, 155-158 (1997). For example, Aβ42 hasthe sequence:

(SEQ ID NO: 33) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT

Aβ41, Aβ40 and Aβ39 differ from Aβ42 by the omission of Ala, Ala-Ile,and Ala-Ile-Val respectively from the C-terminal end. Aβ43 differs fromAβ42 by the presence of a Thr residue at the C-terminus.

Analogous agents to those described above for alpha-SN have previouslybeen described for Aβ (see WO 98/25386 and WO 00/72880, both of whichare incorporated herein for all purposes). These agents include Aβ andactive fragments thereof, conjugates of Aβ, and conjugates of Aβ activefragments, antibodies to Aβ and active fragments thereof (e.g., mouse,humanized, human, and chimeric antibodies), and nucleic acids encodingantibody chains. Active fragments from the N-terminal half of Aβ arepreferred. Preferred immunogenic fragments include Aβ1-5, 1-6, 1-7,1-10, 3-7, 1-3; and 1-4. The designation Aβ1-5 for example, indicates afragment including residues 1-5 of Aβ and lacking other residues of Aβ.Fragments beginning at residues 1-3 of Aβ and ending at residues 7-11 ofAβ are particularly preferred.

The disclosures herein which relates to agents inducing an active immuneresponse, agents for inducing a passive immune response, conjugates, andnucleic acids encoding therapeutic agents (see Sections II. 1, 2, 3, and4, above) apply mutatis mutandis to the use of Aβ and fragments thereof.The disclosures herein which relate to agents inducing an active immuneresponse, agents for inducing a passive immune response, conjugates, andnucleic acids encoding therapeutic agents (see Sections II. 1, 2, 3, and4, above) apply mutatis mutandis to the use of Aβ and fragments thereof.The disclosures herein which relate to patients amendable to treatment,and treatment regimes (see Sections IV and V, below) apply mutatismutandis to the use of Aβ and fragments thereof.

Disaggregated Aβ or fragments thereof means monomeric peptide units.Disaggregated Aβ or fragments thereof are generally soluble, and arecapable of self-aggregating to form soluble oligomers. Oligomers of Aβand fragments thereof are usually soluble and exist predominantly asalpha-helices or random coils. Aggregated Aβ or fragments thereof, meansoligomers of alpha-SN or fragments thereof that have associate intoinsoluble beta-sheet assemblies. Aggregated Aβ or fragments thereof,means also means fibrillar polymers. Fibrils are usually insoluble. Someantibodies bind either soluble Aβ or fragments thereof or aggregated Aβor fragments thereof. Some antibodies bind both soluble Aβ or fragmentsthereof and aggregated Aβ or fragments thereof.

Some examples of conjugates include:AN90549 (Aβ1-7-Tetanus toxoid 830-844 in a MAP4 configuration):

DAEFRHD-QYIKANSKFIGITEL (SEQ ID NO: 34)AN90550 (Aβ1-7-Tetanus toxoid 947-967 in a MAP4 configuration):

DAEFRHD-FNNFTVSFWLRVPKVSASHLE  (SEQ ID NO: 35)AN90542 (Aβ1-7-Tetanus toxoid 830-844+947-967 in a linearconfiguration):

(SEQ ID NO: 36) DAEFRHD-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLEAN90576: (Aβ3-9)-Tetanus toxoid 830-844 in a MAP4 configuration):

EFRHDSG-QYIKANSKFIGITEL (SEQ ID NO: 37)

PADRE peptide (all in linear configurations), wherein X is preferablycyclohexylalanine, tyrosine or phenylalanine, with cyclohexylalaninebeing most preferred:

AN90562 (PADRE-Aβ1-7):

AKXVAAWTLAAA-DAEFRHD (SEQ ID NO: 38)

AN90543 (3 PADRE-Aβ1-7):

(SEQ ID NO: 39) DAEFRHD-DAEFRHD-DAEFRHD-AKXVAAWTLKAAA

Other examples of fusion proteins (immunogenic epitope of Aβ bolded)include:

(SEQ ID NO: 40) AKXVAAWTLKAAA-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 41)DAEFRHD-AKXVAAWTLKAAA  (SEQ ID NO: 42) DAEFRHD-ISQAVHAAHAEINEAGR (SEQ ID NO: 43) FRHDSGY-ISQAVHAAHAEINEAGR  (SEQ ID NO: 44)EFRHDSG-ISQAVHAAHAEINEAGR  (SEQ ID NO: 45)PKYVKQNTLKLAT-DAEFRHD-DAEFRHD-DAEFRHD (SEQ ID NO: 46)DAEFRHD-PKYVKQNTLKLAT-DAEFRHD  (SEQ ID NO: 47)DAEFRHD-DAEFRHD-DAEFRHD-PKYVKQNTLKLAT  (SEQ ID NO: 48)DAEFRHD-DAEFRHD-PKYVKQNTLKLAT  (SEQ ID NO: 49)DAEFRHD-PKYVKQNTLKLAT-EKKIAKMEKASSVFNV-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE-DAEFRHD (SEQ ID NO: 50)DAEFRHD-DAEFRHD-DAEFRHD-QYIKANSKFIGITELNNFTVSFWLR  VPKVSASHLE(SEQ ID NO: 51) DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 52) DAEFRHD-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-DAE FRHD(SEQ ID NO: 53) DAEFRHD-QYIKANSKFIGITEL on a 2 branched resin.

Preferred monoclonal antibodies bind to an epitope within residues 1-10of Aβ (with the first N terminal residue of natural Aβ designated 1).Some preferred monoclonal antibodies bind to an epitope within aminoacids 1-5, and some to an epitope within 5-10. Some preferred antibodiesbind to epitopes within amino acids 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7. Somepreferred antibodies bind to an epitope starting at resides 1-3 andending at residues 7-11 of Aβ. Other antibodies include those binding toepitopes with residues 13-280 (e.g., monoclonal antibody 266). Preferredantibodies have human IgG1 isotype.

IV. Screening Antibodies for Clearing Activity

The invention provides methods of screening an antibody for activity inclearing a Lewy body or any other antigen, or associated biologicalentity, for which clearing activity is desired. To screen for activityagainst a Lewy body, a tissue sample from a brain of a patient with PDor an animal model having characteristic Parkinson's pathology iscontacted with phagocytic cells bearing an Fc receptor, such asmicroglial cells, and the antibody under test in a medium in vitro. Thephagocytic cells can be a primary culture or a cell line, such as BV-2,C8-B4, or THP-1. In some methods, the components are combined on amicroscope slide to facilitate microscopic monitoring. In some methods,multiple reactions are performed in parallel in the wells of amicrotiter dish. In such a format, a separate miniature microscope slidecan be mounted in the separate wells, or a nonmicroscopic detectionformat, such as ELISA detection of alpha-SN can be used. Preferably, aseries of measurements is made of the amount of Lewy body in the invitro reaction mixture, starting from a baseline value before thereaction has proceeded, and one or more test values during the reaction.The antigen can be detected by staining, for example, with afluorescently labeled antibody to alpha-SN or other component of amyloidplaques. The antibody used for staining may or may not be the same asthe antibody being tested for clearing activity. A reduction relative tobaseline during the reaction of the LBs indicates that the antibodyunder test has clearing activity. Such antibodies are likely to beuseful in preventing or treating PD and other LBD.

Analogous methods can be used to screen antibodies for activity inclearing other types of biological entities. The assay can be used todetect clearing activity against virtually any kind of biologicalentity. Typically, the biological entity has some role in human oranimal disease. The biological entity can be provided as a tissue sampleor in isolated form. If provided as a tissue sample, the tissue sampleis preferably unfixed to allow ready access to components of the tissuesample and to avoid perturbing the conformation of the componentsincidental to fixing. Examples of tissue samples that can be tested inthis assay include cancerous tissue, precancerous tissue, tissuecontaining benign growths such as warts or moles, tissue infected withpathogenic microorganisms, tissue infiltrated with inflammatory cells,tissue bearing pathological matrices between cells (e.g., fibrinouspericarditis), tissue bearing aberrant antigens, and scar tissue.Examples of isolated biological entities that can be used includealpha-SN, viral antigens or viruses, proteoglycans, antigens of otherpathogenic microorganisms, tumor antigens, and adhesion molecules. Suchantigens can be obtained from natural sources, recombinant expression orchemical synthesis, among other means. The tissue sample or isolatedbiological entity is contacted with phagocytic cells bearing Fcreceptors, such as monocytes or microglial cells, and an antibody to betested in a medium. The antibody can be directed to the biologicalentity under test or to an antigen associated with the entity. In thelatter situation, the object is to test whether the biological entity isvicariously phagocytosed with the antigen. Usually, although notnecessarily, the antibody and biological entity (sometimes with anassociated antigen) are contacted with each other before adding thephagocytic cells. The concentration of the biological entity and/or theassociated antigen, if present, remaining in the medium is thenmonitored. A reduction in the amount or concentration of antigen or theassociated biological entity in the medium indicates the antibody has aclearing response against the antigen and/or associated biologicalentity in conjunction with the phagocytic cells.

Antibodies or other agents can also be screened for activity in clearingLewy bodies using the in vitro assay described in Example II. Neuronalcells transfected with an expression vector expressing synuclein formsynuclein inclusions that can be visualized microscopically. Theactivity of an antibody or other agent in clearing such inclusions canbe determined comparing appearance or level of synuclein in transfectedcells treated with agent with appearance or level of synuclein incontrol cells not treated with the agent. A reduction in size orintensity of synuclein inclusions or a reduction in level of synucleinsignals activity in clearing synuclein. The activity can be monitoredeither by visualizing synuclein inclusions microscopically or by runningcell extracts on a gel and visualizing a synuclein band. As noted inExample 1, section 2, the change in level of synuclein is most marked ifthe extracts are fractionated into cytosolic and membrane fractions, andthe membrane fraction is analyzed.

V. Patients Amenable to Treatment

Patients amenable to treatment include individuals at risk of asynucleinopathic disease but not showing symptoms, as well as patientspresently showing symptoms. Patients amenable to treatment also includeindividuals at risk of disease of a LBD but not showing symptoms, aswell as patients presently showing symptoms. Such diseases includeParkinson's disease (including idiopathic Parkinson's disease), DLB,DLBD, LBVAD, pure autonomic failure, Lewy body dysphagia, incidentalLBD, inherited LBD (e.g., mutations of the alpha-SN gene, PARK3 andPARK4) and multiple system atrophy (e.g., olivopontocerebellar atrophy,striatonigral degeneration and Shy-Drager syndrome). Therefore, thepresent methods can be administered prophylactically to individuals whohave a known genetic risk of a LBD. Such individuals include thosehaving relatives who have experienced this disease, and those whose riskis determined by analysis of genetic or biochemical markers. Geneticmarkers of risk toward PD include mutations in the synuclein or Parkin,UCHLI, and CYP2D6 genes; particularly mutations at position 53 of thesynuclein gene. Individuals presently suffering from Parkinson's diseasecan be recognized from its clinical manifestations including restingtremor, muscular rigidity, bradykinesia and postural instability.

In some methods, is free of clinical symptoms, signs and/or risk factorsof any amyloidogenic disease and suffers from at least onesynucleinopathic disease. In some methods, the patient is free ofclinical symptoms, signs and/or risk factors of any diseasecharacterized by extracellular amyloid deposits. In some methods, thepatient is free of diseases characterized by amyloid deposits of Aβpeptide. In some methods, the patient is free of clinical symptoms,signs and/or risk factors of Alzheimer's disease. In some methods, thepatient is free of clinical symptoms, signs and/or risk factors ofAlzheimer's disease, cognitive impairment, mild cognitive impairment andDown's syndrome. In some methods, the patient has concurrent Alzheimer'sdisease and a disease characterized by Lewy bodies. In some methods, thepatient has concurrent Alzheimer's disease and a disease characterizedsynuclein accumulation. In some methods, the patient has concurrentAlzheimer's and Parkinson's disease.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,or 30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60, or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody, or activated T-cell or B-cell responses to the therapeuticagent (e.g., alpha-SN peptide or Aβ, or both) over time. If the responsefalls, a booster dosage is indicated.

Optionally, presence of absence of symptoms, signs or risk factors of adisease is determined before beginning treatment.

Vi. Treatment Regimes

In general treatment regimes involve administering an agent effective toinduce an immunogenic response to alpha-SN and/or an agent effective toinduce an immunogenic response to Aβ to a patient. In prophylacticapplications, pharmaceutical compositions or medicaments areadministered to a patient susceptible to, or otherwise at risk of a LBDin regime comprising an amount and frequency of administration of thecomposition or medicament sufficient to eliminate or reduce the risk,lessen the severity, or delay the outset of the disease, includingphysiological, biochemical, histologic and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. In therapeuticapplications, compositions or medicates are administered to a patientsuspected of, or already suffering from such a disease in a regimecomprising an amount and frequency of administration of the compositionsufficient to cure, or at least partially arrest, the symptoms of thedisease (physiological, biochemical, histologic and/or behavioral),including its complications and intermediate pathological phenotypes indevelopment of the disease. An amount adequate to accomplish therapeuticor prophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. A combination of amount and dosagefrequency adequate to accomplish therapeutic or prophylactic treatmentis defined as a therapeutically or prophylactically-effective regime. Inboth prophylactic and therapeutic regimes, agents are usuallyadministered in several dosages until a sufficient immune response hasbeen achieved. Typically, the immune response is monitored and repeateddosages are given if the immune response starts to wane.

In some methods, administration of an agent results in reduction ofintracellular levels of aggregated synuclein. In some methods,administration of an agent results in improvement in a clinical symptomof a LBD, such as motor function in the case of Parkinson's disease. Insome methods, reduction in intracellular levels of aggregated synucleinor improvement in a clinical symptom of disease is monitored atintervals after administration of an agent.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnonhuman mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.The amount of immunogen depends on whether adjuvant is alsoadministered, with higher dosages being required in the absence ofadjuvant. The amount of an immunogen for administration sometimes variesfrom 1-500 μg per patient and more usually from 5-500 μg per injectionfor human administration. Occasionally, a higher dose of 1-2 mg perinjection is used. Typically about 10, 20, 50 or 100 μg is used for eachhuman injection. The mass of immunogen also depends on the mass ratio ofimmunogenic epitope within the immunogen to the mass of immunogen as awhole. Typically, 10⁻³ to 10⁻⁵ micromoles of immunogenic epitope areused for microgram of immunogen. The timing of injections can varysignificantly from once a day, to once a year, to once a decade. On anygiven day that a dosage of immunogen is given, the dosage is greaterthan 1 μg/patient and usually greater than 10 μg/patient if adjuvant isalso administered, and greater than 10 μg/patient and usually greaterthan 100 μg/patient in the absence of adjuvant. A typical regimenconsists of an immunization followed by booster injections at timeintervals, such as 6 week intervals. Another regimen consists of animmunization followed by booster injections 1, 2 and 12 months later.Another regimen entails an injection every two months for life.Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg or, in other words, 70 mgs or700 mgs or within the range of 70-700 mgs, respectively, for a 70 kgpatient. An exemplary treatment regime entails administration once perevery two weeks or once a month or once every 3 to 6 months. In somemethods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toalpha-SN in the patient. In some methods, dosage is adjusted to achievea plasma antibody concentration of 1-1000 ug/ml and in some methods25-300 ug/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. In general, human antibodies show the longesthalf life, followed by humanized antibodies, chimeric antibodies, andnonhuman antibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patent canbe administered a prophylactic regime.

Doses for nucleic acids encoding immunogens range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intrathecal, intraperitoneal, intranasal or intramuscularmeans for prophylactic and/or therapeutic treatment. The most typicalroute of administration of an immunogenic agent is subcutaneous althoughother routes can be equally effective. The next most common route isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. In some methods, agents areinjected directly into a particular tissue where deposits haveaccumulated, for example intracranial injection. Intramuscular injectionor intravenous infusion are preferred for administration of antibody. Insome methods, particular therapeutic antibodies are injected directlyinto the cranium. In some methods, antibodies are administered as asustained release composition or device, such as a Medipad™ device.

As noted above, agents inducing an immunogenic response against alpha-SNand Aβ respectively can be administered in combination. The agents canbe combined in a single preparation or kit for simultaneous, sequentialor separate use. The agents can occupy separate vials in the preparationor kit or can be combined in a single vial. These agents of theinvention can optionally be administered in combination with otheragents that are at least partly effective in treatment of LBD. In thecase of Parkinson's Disease and Down's syndrome, in which LBs occur inthe brain, agents of the invention can also be administered inconjunction with other agents that increase passage of the agents of theinvention across the blood-brain barrier.

Immunogenic agents of the invention, such as peptides, are sometimesadministered in combination with an adjuvant. A variety of adjuvants canbe used in combination with a peptide, such as alpha-SN, to elicit animmune response. Preferred adjuvants augment the intrinsic response toan immunogen without causing conformational changes in the immunogenthat affect the qualitative form of the response. Preferred adjuvantsinclude aluminum hydroxide and aluminum phosphate, 3 De-O-acylatedmonophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem ResearchInc., Hamilton, Mont., now part of Corixa). Stimulon™ QS-21 is atriterpene glycoside or saponin isolated from the bark of the QuillajaSaponaria Molina tree found in South America (see Kensil et al., inVaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman,Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540), (AquilaBioPharmaceuticals, Framingham, Mass.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killedmycobacteria. Another adjuvant is CpG (WO 98/40100). Alternatively,alpha-SN or Aβ can be coupled to an adjuvant. However, such couplingshould not substantially change the conformation of alpha-SN so as toaffect the nature of the immune response thereto. Adjuvants can beadministered as a component of a therapeutic composition with an activeagent or can be administered separately, before, concurrently with, orafter administration of the therapeutic agent.

A preferred class of adjuvants is aluminum salts (alum), such as alumhydroxide, alum phosphate, alum sulfate. Such adjuvants can be used withor without other specific immunostimulating agents such as MPL or 3-DMP,QS-21, polymeric or monomeric amino acids such as polyglutamic acid orpolylysine. Another class of adjuvants is oil-in-water emulsionformulations. Such adjuvants can be used with or without other specificimmunostimulating agents such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi ImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™).

Another class of preferred adjuvants is saponin adjuvants, such asStimulon™ (QS-21, Aquila, Framingham, Mass.) or particles generatedtherefrom such as ISCOMs (immunostimulating complexes) and ISCOMATRIX.Other adjuvants include RC-529, GM-CSF and Complete Freund's Adjuvant(CFA) and Incomplete Freund's Adjuvant (IFA). Other adjuvants includecytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-6, IL-12,IL13, and IL-15), macrophage colony stimulating factor (M-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), and tumornecrosis factor (TNF). Another class of adjuvants is glycolipidanalogues including N-glycosylamides, N-glycosylureas andN-glycosylcarbamates, each of which is substituted in the sugar residueby an amino acid, as immuno-modulators or adjuvants (see U.S. Pat. No.4,855,283). Heat shock proteins, e.g., HSP70 and HSP90, may also be usedas adjuvants.

An adjuvant can be administered with an immunogen as a singlecomposition, or can be administered before, concurrent with or afteradministration of the immunogen. Immunogen and adjuvant can be packagedand supplied in the same vial or can be packaged in separate vials andmixed before use. Immunogen and adjuvant are typically packaged with alabel indicating the intended therapeutic application. If immunogen andadjuvant are packaged separately, the packaging typically includesinstructions for mixing before use. The choice of an adjuvant and/orcarrier depends on the stability of the immunogenic formulationcontaining the adjuvant, the route of administration, the dosingschedule, the efficacy of the adjuvant for the species being vaccinated,and, in humans, a pharmaceutically acceptable adjuvant is one that hasbeen approved or is approvable for human administration by pertinentregulatory bodies. For example, Complete Freund's adjuvant is notsuitable for human administration. Alum, MPL and QS-21 are preferred.Optionally, two or more different adjuvants can be used simultaneously.Preferred combinations include alum with MPL, alum with QS-21, MPL withQS-21, MPL or RC-529 with GM-CSF, and alum, QS-21 and MPL together.Also, Incomplete Freund's adjuvant can be used (Chang et al., AdvancedDrug Delivery Reviews 32, 173-186 (1998)), optionally in combinationwith any of alum, QS-21, and MPL and all combinations thereof.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980). The preferred form depends on the intended mode of administrationand therapeutic application. The compositions can also include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The diluent is selected so as not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl. Compositions for parenteral administration aretypically substantially sterile, substantially isotonic and manufacturedunder GMP conditions of the FDA or similar body. For example,compositions containing biologics are typically sterilized by filtersterilization. Compositions can be formulated for single doseadministration.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249,1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997).The agents of this invention can be administered in the form of a depotinjection or implant preparation which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications.

For suppositories, binders and carriers include, for example,polyalkylene glycols or triglycerides; such suppositories can be formedfrom mixtures containing the active ingredient in the range of 0.5% to10%, preferably 1%-2%. Oral formulations include excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, and magnesium carbonate. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10%-95%of active ingredient, preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995);Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).

VII. Methods of Monitoring and Methods of Diagnosis

The invention provides methods of detecting an immune response againstalpha-SN peptide and/or Aβ peptide in a patient suffering from orsusceptible to a LBD. The methods are particularly useful for monitoringa course of treatment being administered to a patient. The methods canbe used to monitor both therapeutic treatment on symptomatic patientsand prophylactic treatment on asymptomatic patients. The methods areuseful for monitoring both active immunization (e.g., antibody producedin response to administration of immunogen) and passive immunization(e.g., measuring level of administered antibody).

1. Active Immunization

Some methods entail determining a baseline value of an immune responsein a patient before administering a dosage of agent, and comparing thiswith a value for the immune response after treatment. A significantincrease (i.e., greater than the typical margin of experimental error inrepeat measurements of the same sample, expressed as one standarddeviation from the mean of such measurements) in value of the immuneresponse signals a positive treatment outcome (i.e., that administrationof the agent has achieved or augmented an immune response). If the valuefor immune response does not change significantly, or decreases, anegative treatment outcome is indicated. In general, patients undergoingan initial course of treatment with an immunogenic agent are expected toshow an increase in immune response with successive dosages, whicheventually reaches a plateau. Administration of agent is generallycontinued while the immune response is increasing. Attainment of theplateau is an indicator that the administered of treatment can bediscontinued or reduced in dosage or frequency.

In other methods, a control value (i.e., a mean and standard deviation)of immune response is determined for a control population. Typically theindividuals in the control population have not received prior treatment.Measured values of immune response in a patient after administering atherapeutic agent are then compared with the control value. Asignificant increase relative to the control value (e.g., greater thanone standard deviation from the mean) signals a positive treatmentoutcome. A lack of significant increase or a decrease signals a negativetreatment outcome. Administration of agent is generally continued whilethe immune response is increasing relative to the control value. Asbefore, attainment of a plateau relative to control values in anindicator that the administration of treatment can be discontinued orreduced in dosage or frequency.

In other methods, a control value of immune response (e.g., a mean andstandard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose immune responses have reached a plateau in response to treatment.Measured values of immune response in a patient are compared with thecontrol value. If the measured level in a patient is not significantlydifferent (e.g., more than one standard deviation) from the controlvalue, treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment regime, for example, use of adifferent adjuvant may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for immuneresponse to determine whether a resumption of treatment is required. Themeasured value of immune response in the patient can be compared with avalue of immune response previously achieved in the patient after aprevious course of treatment. A significant decrease relative to theprevious measurement (i.e., greater than a typical margin of error inrepeat measurements of the same sample) is an indication that treatmentcan be resumed. Alternatively, the value measured in a patient can becompared with a control value (mean plus standard deviation) determinedin a population of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousor cerebrospinal fluid from the patient. The sample is analyzed forindication of an immune response to any form of alpha-SN, typically NAC,or Aβ. The immune response can be determined from the presence of, e.g.,antibodies or T-cells that specifically bind to alpha-SN or AB. ELISAmethods of detecting antibodies specific to alpha-SN are described inthe Examples section. Methods of detecting reactive T-cells have beendescribed above (see Definitions). In some methods, the immune responseis determined using a clearing assay, such as described in Section IIIabove. In such methods, a tissue or blood sample from a patient beingtested is contacted with LBs (e.g., from a synuclein/hAPP transgenicmouse) and phagocytic cells bearing Fc receptors. Subsequent clearing ofthe LBs is then monitored. The existence and extent of clearing responseprovides an indication of the existence and level of antibodieseffective to clear alpha-SN in the tissue sample of the patient undertest.

2. Passive Immunization

In general, the procedures for monitoring passive immunization aresimilar to those for monitoring active immunization described above.However, the antibody profile following passive immunization typicallyshows an immediate peak in antibody concentration followed by anexponential decay. Without a further dosage, the decay approachespretreatment levels within a period of days to months depending on thehalf-life of the antibody administered. For example the half-life ofsome human antibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to alpha-SN in thepatient is made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

3. Diagnostic Kits

The invention further provides diagnostic kits for performing thediagnostic methods described above. Typically, such kits contain anagent that specifically binds to antibodies to alpha-SN. The kit canalso include a label. For detection of antibodies to alpha-SN, the labelis typically in the form of labeled anti-idiotypic antibodies. Fordetection of antibodies, the agent can be supplied prebound to a solidphase, such as to the wells of a microtiter dish. Kits also typicallycontain labeling providing directions for use of the kit. The labelingmay also include a chart or other correspondence regime correlatinglevels of measured label with levels of antibodies to alpha-SN. The termlabeling refers to any written or recorded material that is attached to,or otherwise accompanies a kit at any time during its manufacture,transport, sale or use. For example, the term labeling encompassesadvertising leaflets and brochures, packaging materials, instructions,audio or video cassettes, computer discs, as well as writing imprinteddirectly on kits.

The invention also provides diagnostic kits for performing in vivoimaging. Such kits typically contain an antibody binding to an epitopeof alpha-SN, preferably within NAC. Preferably, the antibody is labeledor a secondary labeling reagent is included in the kit. Preferably, thekit is labeled with instructions for performing an in vivo imagingassay.

VIII. In Vivo Imaging

The invention provides methods of in vivo imaging LBs in a patient. Suchmethods are useful to diagnose or confirm diagnosis of PD, or otherdisease associated with the presence of LBs in the brain, orsusceptibility thereto. For example, the methods can be used on apatient presenting with symptoms of dementia. If the patient has LBs,then the patient is likely suffering from, e.g. PD. The methods can alsobe used on asymptomatic patients. Presence of abnormal deposits ofamyloid indicates susceptibility to future symptomatic disease. Themethods are also useful for monitoring disease progression and/orresponse to treatment in patients who have been previously diagnosedwith Parkinson's disease.

The methods work by administering a reagent, such as antibody that bindsto alpha-SN in the patient and then detecting the agent after it hasbound. Preferred antibodies bind to alpha-SN deposits in a patientwithout binding to full length NACP polypeptide. Antibodies binding toan epitope of alpha-SN within NAC are particularly preferred. Ifdesired, the clearing response can be avoided by using antibodyfragments lacking a full length constant region, such as Fabs. In somemethods, the same antibody can serve as both a treatment and diagnosticreagent. In general, antibodies binding to epitopes N-terminal ofalpha-SN do not show as strong signal as antibodies binding to epitopesC-terminal, presumably because the N-terminal epitopes are inaccessiblein LBs (Spillantini et al PNAS, 1998). Accordingly, such antibodies areless preferred.

Diagnostic reagents can be administered by intravenous injection intothe body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labeled, although in some methods, the primary reagentwith affinity for alpha-SN is unlabelled and a secondary labeling agentis used to bind to the primary reagent. The choice of label depends onthe means of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size and/or intensity oflabeled loci to corresponding base line values. The base line values canrepresent the mean levels in a population of undiseased individuals.Base line values can also represent previous levels determined in thesame patient. For example, base line values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the base line values. A decrease in values relative tobase line signals a positive response to treatment.

EXAMPLES I. Immunization of Human Alpha-Synuclein Transgenic Mice withHuman Alpha-Synuclein Results in the Production of High TiterAnti-Alpha-Synuclein Antibodies that Cross the Blood-Brain Barrier

Full-length recombinant human alpha-SN was resuspended at aconcentration of 1 mg/ml in 1× phosphate buffered saline (PBS). For eachinjection, 50 μl of alpha-SN was used; giving a final concentration of50 μg per injection to which 150 μl of 1×PBS was added. CompleteFreund's adjuvant (CFA) was then added 1:1 to either alpha-SN or PBSalone (control), vortexed and sonicated to completely resuspend theemulsion. For the initial injections, eight D line human alpha-SNtransgenic (tg) single transgenic 4-7 months old mice (Masliah, et al.Science 287:1265-1269 (2000) received injections of human alpha-SN inCFA and, as control, four D line human alpha-SN tg mice receivedinjections of PBS in CFA. Mice received a total of 6 injections. Threeinjections were performed at two weeks intervals and then 3 injectionsat one month intervals. Animals were sacrificed using NIH Guidelines forthe humane treatment of animals 5 months after initiation of theexperiment. After blood samples were collected for determination ofantibody titers, brains were immersion-fixed for 4 days in 4%paraformaldehyde in PBS. Levels of antibodies against human alpha-SN byELISA are shown in Table 1. The treated mice are divided into two groupsby titer. The first group developed a moderate titer of 2-8,000. Thesecond group developed a high titer of 12000-30000. No titer was foundin control mice. Neuropathological analysis showed that mice producinghigh titers had a marked decrease in the size of synuclein incusions.Mice producing moderate titers showed a smaller decrease. FIG. 2 (panelsa-d) show synuclein inclusions in (a) a nontransgenic mouse, (b) atransgenic mouse treated with CFA only, (c) a transgenic mouse immunizedwith alpha synuclein and CFA that developed a moderate titer and (d) atransgenic mouse immunized with alpha synuclein and CFA that developed ahigher titer. Samples were visualized by immunostaining with ananti-human alpha-SN antibody. FIG. 2 shows synuclein inclusions in panel(b) but not panel (a). In panel (c), treated mouse, moderate titers, theinclusions are somewhat reduced in intensity. In panel (d) theinclusions are markedly reduced in intensity. Panels (e)-(h) show levelsof anti-IgG in the brains same four mice as panels (a) to (d)respectively. It can be seen that IgG is present in panels (g) and to agreater extent in panel (h). The data shows that peripherallyadministered antibodies to alpha-SN cross the blood brain harrier andreach the brain. Panels (i) to (l) showing staining for GAP, a marker ofastroglial cells, again for the same four mice as in the first two rowsof the figure. It can be seen that panels (k) and (l) show moderatelyincreased staining compared with (i) and (j). These data show thatclearing of synuclein deposits is accompanied by a mild astroglial andmicroglial reaction.

TABLE 1 Syn (+) Group Genotype n= Age at Sac Treatment/Length Titersinclusions/mm2 I Syn Tg 4 10-13 mo a-syn + CFA 2,000-8,000 15-29 50ug/inj for 3 mo sac'd 3 mo later II Syn Tg 4 10-13 mo a-syn + CFA12,000-30,000 10-22 50 ug/inj for 3 mo sac'd 3 mo later III Syn Tg 410-13 mo PBS + CFA for 0 18-29 3 mo sac'd 3 mo later

II. In Vitro Screen for Antibodies Clearing Synuclein Inclusions

GT1-7 neuronal cell (Hsue et al. Am. J. Pathol. 157:401-410 (2000)) weretransfected with a pCR3.1-T expression vector (Invitrogen, Carlsbad,Calif.) expressing murine alpha-SN and compared with cells transfectedwith expression vector alone (FIG. 3, panels B and A respectively).Cells transfected with vector alone (panel A) have a fibroblasticappearance while cells transfected with alpha-SN are rounded, withinclusion bodies at the cell surface visible via both light and confocalscanning microscopy. Transfected cells were then treated with rabbitpreimmune serum (panel C) or 67-10, an affinity purified rabbitpolyclonal antibody against a murine alpha-SN C terminal residues131-140 (Iwai, et al., Neuron 14:467 (1995) (panel D). It can be seenthat the inclusion bodies stain less strongly in panel D than in panel Cindicating that the antibody against alpha synuclein was effective inclearing or preventing the development of inclusions. FIG. 4 shows a gelanalysis of particulate and cytosolic fractions of GT1-7 transfectedcells treated with the rabbit preimmune serum and 67-10 polyclonalantibody. It can be seen that the synuclein levels in the cytosolicfraction is largely unchanged by treatment with preimmune serum orantibody to alpha-SN. However, the alpha-SN band disappears in themembrane fraction of GT1-7 cells treated with antibody to alpha-SN.These data indicates that the alpha synuclein antibody activity resultsin the clearance of synuclein associated with the cellular membrane.

Transfected GT1-7 cells can be used to screen antibodies for activity inclearing synuclein incusions with detection either byimmunohistochemical analysis, light microscopy as in FIG. 3 or by gelanalysis as in FIG. 4.

III. Prophylactic and Therapeutic Efficacy of Immunization withAlpha-Synuclein

i. Immunization of Human Alpha-Synuclein tg Mice

For this study, heterozygous human alpha-SN transgenic (tg) mice (LineD) (Masliah et al., Am. J. Pathol (1996) 148:201-10) and nontransgenic(nontg) controls are used. Experimental animals are divided into 3groups. For group I, the preventive effects of early immunization byimmunizing mice for 8 months beginning at 2 months of age are tested.For group II, young adult mice are vaccinated for 8 months beginning atthe age of 6 months to determine whether immunization can reduce diseaseprogression once moderate pathology had been established. For group III,older mice are immunized for 4 months beginning at the age of 12 monthsto determine whether immunization can reduce the severity of symptomsonce robust pathology has been established. For all groups, mice areimmunized with either recombinant human alpha-SN plus CFA or CFA alone,and for each experiment 20 tg and 10 nontg mice are used. Of them, 10 tgmice are immunized with human alpha-SN+CFA and other 10 tg with CFAalone. Similarly, 5 nontg mice are immunized with human alpha-SN+CFA andthe other 5 with CFA alone. Briefly, the immunization protocol consistsof an initial injection with purified recombinant human alpha-SN (2mg/ml) in CFA, followed by a reinjection 1 month later with humanalpha-SN in combination with IFA. Mice are then re-injected with thismixture once a month. In a small subset of human alpha-SN tg (n=3/each;6-months-old) and nontg (n=3/each; 6-month-old) mice, additionalexperiments consisting of immunization with murine (m) alpha-SN, humanbeta synuclein or mutant (A53T) human alpha-SN are performed.

Levels of alpha-SN antibody are determined using 96-well microtiterplates coated with 0.4 μg per well of purified full-length alpha-SN byovernight incubation at 4° C. in sodium carbonate buffer, pH 9.6. Wellsare washed 4× with 200 μl each PBS containing 0.1% Tween and blocked for1 hour in PBS-1% BSA at 37° C. Serum samples are serially diluted“in-well”, 1:3, starting in row A, ranging from a 1:150 to 1:328,050dilution. For control experiments, a sample of mouse monoclonal antibodyis run against alpha-SN, no protein, and buffer-only blanks. The samplesare incubated overnight at 4° C. followed by a 2-hour incubation withgoat anti-mouse IgG alkaline phosphatase-conjugated antibody (1:7500,Promega, Madison, Wis.). Atto-phos® alkaline phophatase fluorescentsubstrate is then added for 30 minutes at room temperature. The plate isread at an excitation wavelength of 450 nm and an emission wavelength of550 nm. Results are plotted on a semi-log graph with relativefluorescence units on the ordinate and serum dilution on the abscissa.Antibody titer is defined as the dilution at which there was a 50%reduction from maximal antibody binding.

For each group, at the end of the treatment, mice undergo motorassessment in the rotarod, as described (Masliah, et al. (2000)). Afteranalysis, mice are euthanized and brains are removed for detailedneurochemical and neuropathological analysis as described below.Briefly, the right hemibrain is frozen and homogenized fordeterminations of aggregated and non-aggregated human alpha-SNimmunoreactivity by Western blot (Masliah, et al. (2000)). The lefthemibrain is fixed in 4% paraformaldehyde, serially sectioned in thevibratome for immunocytochemistry and ultrastructural analysis.

ii. Immunocytochemical and Neuropathological Analysis.

In order to determine if immunization decreases, human alpha-SNaggregation sections are immunostained with a rabbit polyclonal antibodyagainst human alpha-SN (1:500). After an overnight incubation at 4° C.,sections are incubated with biotinylated anti-rabbit secondary antibodyfollowed by Avidin D-Horseradish peroxidase (HRP) complex (1:200, ABCElite, Vector). Sections are also immunostained with biotinylatedanti-rabbit, mouse or human secondary alone. The experiments with theanti-mouse secondary determine whether the antibodies against humanalpha-SN cross into the brain. The reaction is visualized with 0.1%3,3,-diaminobenzidine tetrahydrochloride (DAB) in 50 mM Tris-HCl (pH7.4) with 0.001% H₂O₂ and sections are then mounted on slided underEntellan. Levels of immunoreactivity are semiquantitatively assessed byoptical densitometry using the Quantimet 570C. These sections are alsostudied by image analysis to determine the numbers of alpha-SNimmunoractive inclusions and this reliable measure of alpha-SNaggregation acts as a valuable index of the anti-aggregation effects ofvaccination (Masliah, et al. (2000)).

Analysis of patterns of neurodegeneration is achieved by analyzingsynaptic and dendritic densities in the hippocampus, frontal cortex,temporal cortex and basal ganglia utilizing vibratome sectionsdouble-immunolabeled for synaptophysin and microtubule-associatedprotein 2 (MAP2) and visualized with LSCM. Additional analysis ofneurodegeneration is achieved by determining tyrosine hydroxylase (TH)immunoreactivity in the caudoputamen and substantia nigra (SN) aspreviously described (Masliah, et al. (2000)). Sections will be imagedwith the LSCM and each individual image is interactively thresholdedsuch that the TH-immunoreactive terminals displaying pixel intensitywithin a linear range are included. A scale is set to determine thepixel to μm ratio. Then, this information is used to calculate the %area of the neuropil covered by TH-immunoractive terminals. These samesections are also utilized to evaluate the numbers of TH neurons in theSN.

To assess the patterns of immune response to immunization,immunocytochemical and ultrastructural analysis with antibodies againsthuman GFAP, MCH class II, Mac 1, TNF-alpha, IL1beta and IL6 areperformed in the brain sections of nontg and alpha-SN tg mice immunizedwith recombinant human alpha-SN and control immunogens.

iii. Behavioral Analysis.

For locomotor activity mice are analyzed for 2 days in the rotarod (SanDiego) Instruments, San Diego, Calif.), as previously described(Masliah, et al. (2000)). On the first day mice are trained for 5trials: the first one at 10 rpm, the second at 20 rpm and the third tofifth at 40 rpm. On the second day, mice are tested for 7 trials at 40rpm each. Mice are placed individually on the cylinder and the speed ofrotation is increased from 0 to 40 rpm over a period of 240 sec. Thelength of time mice remain on the rod (fall Latency) is recorded andused as a measure of motor function.

IV. Immunization with Alpha-Synuclein Fragments

Human alpha-SN transgenic mice 10-13 months of age are immunized with 9different regions of alpha-SN to determine which epitopes convey theefficacious response. The 9 different immunogens and one control areinjected i.p. as described above. The immunogens include four humanalpha-SN peptide conjugates, all coupled to sheep anti-mouse IgG via acystine link. Alpha-SN and PBS are used as positive and negativecontrols, respectively. Titers are monitored as above and mice areeuthanized at the end of 3-12 months of injections. Histochemistry,alpha-SN levels, and toxicology analysis is determined post mortem.

i. Preparation of Immunogens

Preparation of coupled alpha-SN peptides: H alpha-SN peptide conjugatesare prepared by coupling through an artificial cysteine added to thealpha-SN peptide using the crosslinking reagent sulfo-EMCS. The alpha-SNpeptide derivatives are synthesized with the following final amino acidsequences. In each case, the location of the inserted cysteine residueis indicated by underlining.

alpha-synuclein 60-72 (NAC region) peptide:

NH2-KEQVTNVCGGAVVT-COOH (SEQ ID NO: 54)

alpha-synuclein 73-84 (NAC region) peptide:

NH2-GVTAVAQKTVECG-COOH (SEQ ID NO: 55)

alpha-synuclein 102-112 peptide:

(SEQ ID NO: 56) NH2-C-amino-heptanoic acid-KNEEGAPCQEG-COOH

alpha-synuclein 128-140 peptide:

Ac-NH-PSEEGYQDYEPECA-COOH (SEQ ID NO: 57)

To prepare for the coupling reaction, ten mg of sheep anti-mouse IgG(Jackson ImmunoResearch Laboratories) is dialyzed overnight against 10mM sodium borate buffer, pH 8.5. The dialyzed antibody is thenconcentrated to a volume of 2 mL using an Amicon Centriprep tube. Ten mgsulfo-EMCS

[N (ε-maleimidocuproyloxy) succinimide] (Molecular Sciences Co.) isdissolved in one mL deionized water. A 40-fold molar excess ofsulfo-EMCS is added drop wise with stirring to the sheep anti-mouse IgGand then the solution is stirred for an additional ten min. Theactivated sheep anti-mouse IgG is purified and buffer exchanged bypassage over a 10 mL gel filtration column (Pierce Presto Column,obtained from Pierce Chemicals) equilibrated with 0.1 M NaPO4, 5 mMEDTA, pH 6.5. Antibody containing fractions, identified by absorbance at280 nm, are pooled and diluted to a concentration of approximately 1mg/mL, using 1.4 mg per OD as the extinction coefficient. A 40-foldmolar excess of alpha-SN peptide is dissolved in 20 mL of 10 mM NaPO4,pH 8.0, with the exception of the alpha-SN peptide for which 10 mg isfirst dissolved in 0.5 mL of DMSO and then diluted to 20 mL with the 10mM NaPO4 buffer. The peptide solutions are each added to 10 mL ofactivated sheep anti-mouse IgG and rocked at room temperature for 4 hr.The resulting conjugates are concentrated to a final volume of less than10 mL using an Amicon Centriprep tube and then dialyzed against PBS tobuffer exchange the buffer and remove free peptide. The conjugates arepassed through 0.22 μm-pore size filters for sterilization and thenaliquoted into fractions of 1 mg and stored frozen at −20° C. Theconcentrations of the conjugates are determined using the BCA proteinassay (Pierce Chemicals) with horse IgG for the standard curve.Conjugation is documented by the molecular weight increase of theconjugated peptides relative to that of the activated sheep anti-mouseIgG.

V. Passive Immunization with Antibodies to Alpha-Synuclein

Human alpha-SN mice each are injected with 0.5 mg in PBS ofanti-alpha-SN monoclonals as shown below. All antibody preparations arepurified to have low endotoxin levels. Monoclonals can be preparedagainst a fragment by injecting the fragment or longer form of alpha-SNinto a mouse, preparing hybridomas and screening the hybridomas forantibody that specifically binds to a desired fragment of alpha-SNwithout binding to other nonoverlapping fragments of alpha-SN.

Mice are injected ip as needed over a 4 month period to maintain acirculating antibody concentration measured by ELISA titer of greaterthan 1:1000 defined by ELISA to alpha-SN or other immunogen. Titers aremonitored as above and mice are euthanized at the end of 6 months ofinjections. Histochemistry, alpha-SN levels and toxicology are performedpost mortem.

VI. Aβ Immunization of Syn/APP Transgenic Mice

This experiment compares the effects of Aβ immunization on three typesof transgenic mice: transgenic mice with an alpha synuclein transgene(SYN), APP mice with an APP transgene (Games et al.) and doubletransgenic SYN/APP mice produced by crossing the single transgenic. Thedouble transgenic mice are described in Masliah et al., PNAS USA98:12245-12250 (2001). These mice represent a model of individualshaving both Alzheimer's and Parkinson's disease. Table 2 shows thedifferent groups, the age of the mice used in the study, the treatmentprocedure and the titer of antibodies to Aβ. It can be seen that asignificant titer was generated in all three types of mice. FIG. 5 showsthe % area covered by amyloid plaques of Aβ in the brain determined byexamination of brain sections from treated subjects by microscopy.Substantial deposits accumulate in the APP and SYN/APP mice but not inthe SYN mice or nontransgenic controls. The deposits are greater in theSYN/APP double transgenic mice. Immunization with Aβ1-42 reduces thedeposits in both APP and SYN/APP mice. FIG. 6 shows synuclein depositsin the various groups of mice as detected by confocal laser scanning andlight microscopy. Synuclein deposits accumulate in the SYN and SYN/APPmice treated with CFA only. However, in the same types of mice treatedwith Aβ1-42 and CFA there is a marked reduction in the level ofsynuclein deposit. These data indicate that treatment with Aβ iseffective not only in clearing Aβ deposits but also in clearing depositsof synuclein. Therefore, treatment with Aβ or antibodies thereto isuseful in treating not only Alzheimer's disease but combined Alzheimer'sand Parkinson's disease, and Parkinson's disease in patients free ofAlzheimer's disease. The titer of antiAβ antibodies in SYN/APP micecorrelated with decreased formation of synuclein inclusions (r=−0.71,p<0.01).

TABLE 2 Group n= Age Treatment/Length Ab Titers SYN 4 12-20 mo Ab inj.50 ug/inj 10,000-58,000 for 6 mo SYN 2 12-20 mo Sal inj. for 6 mo 0 APP2 12-20 mo Ab inj. 50 ug/inj 25,000 for 6 mo APP 2 12-20 mo Sal inj. for6 mo 0 SYN/APP 4 12-20 mo Ab inj. 50 ug/inj  1,000-50,000 for 6 moSYN/APP 2 12-20 mo Sal inj. for 6 mo 0

1-70. (canceled)
 71. A method of therapeutically treating a patientsuffering from a Lewy body disease, the method comprising: administeringto the patient an effective regime of an agent and therebytherapeutically treating the disease; wherein (i) the agent is Aβ or animmunogenic fragment thereof and the agent is linked to a carrier thathelps elicit an immune response to the agent or is administered with anadjuvant that augments an immune response to the agent, or (ii) theagent is an antibody to Aβ.
 72. The method of claim 71, wherein theagent is Aβ or an immunogenic fragment thereof.
 73. The method of claim71, wherein the agent is an antibody to Aβ.
 74. The method of claim 71,wherein the agent is administered peripherally.
 75. The method of claim71, wherein the effective regime comprises administering multipledosages over a period of at least six months.
 76. The method of claim71, wherein the administering improves motor characteristics of thepatient.
 77. The method of claim 71, wherein the patient is free ofAlzheimer's disease.
 78. The method of claim 71, wherein the patient isfree of Alzheimer's disease and has no genetic risk factors thereof. 79.A method of therapeutically treating a patient suffering from a Lewybody disease, comprising administering to the patient an effectiveregime of a first agent and a second agent and thereby therapeuticallytreating the disease; wherein (i) the first agent is alpha synuclein oran immunogenic fragment thereof, and the first agent is linked to acarrier that helps elicit an immune response to the first agent or isadministered with an adjuvant that augments an immune response to thefirst agent, or (ii) the first agent is an antibody to alpha synuclein,and wherein (i) the second agent is Aβ or an immunogenic fragmentthereof and the second agent is linked to a carrier that helps elicit animmune response to the second agent or is administered with an adjuvantthat augments an immune response to the second agent, or (ii) the secondagent is an antibody to Aβ.
 80. A method of reducing the risk, lesseningthe severity, or delaying the outset of disease in a patient having aknown genetic risk of a Lewy body disease, the method comprisingadministering to the patient an effective regime of an agent and therebyreducing the risk, lessening the severity, or delaying the outset of thedisease; wherein (i) the agent is Aβ or an immunogenic fragment thereof,and the agent is linked to a carrier that helps elicit an immuneresponse to the agent or is administered with an adjuvant that augmentsan immune response to the agent, or (ii) the agent is an antibody to Aβ.81. The method of claim 80, wherein the agent is Aβ or an immunogenicfragment thereof.
 82. The method of claim 80, wherein the agent is anantibody to Aβ.
 83. A method of reducing the risk, lessening theseverity, or delaying the outset of disease in a patient having a knowngenetic risk of a Lewy body disease, comprising administering to thepatient an effective regime of a first agent and a second agent thatinduces an immunogenic response against Aβ in the patient and therebyreducing the risk, lessening the severity, or delaying the outset of thedisease; wherein (i) the first agent is alpha synuclein or animmunogenic fragment thereof and the first agent is linked to a carrierthat helps elicit an immune response to the first agent or isadministered with an adjuvant that augments an immune response to thefirst agent, or (ii) the first agent is an antibody to alpha synuclein,and wherein (i) the second agent is Aβ or an immunogenic fragmentthereof and the second agent is linked to a carrier that helps elicit animmune response to the second agent or is administered with an adjuvantthat augments an immune response to the second agent, or (ii) the secondagent is an antibody to Aβ.
 84. The method of claim 83, wherein theagent is administered peripherally.
 85. The method of claim 83, whereinthe effective regime comprises administering multiple dosages over aperiod of at least six months.
 86. The method of claim 83, wherein thepatient is free of Alzheimer's disease.
 87. The method of claim 83,wherein the patient is free of Alzheimer's disease and has no geneticrisk factors thereof.